Serine protease variants and polynucleotides encoding same

ABSTRACT

The present invention relates to protease variants, having improved properties compared to the parent protease, in particular variants of a serine protease belonging to family 53 derived from a strain of  Meripilus giganteus . The variants according to the invention have in particular increased thermo-stability, e.g., increased residual activity after 30 min at a temperature in the range from 55 to 60° C. and/or increased thermal denaturation temperature, compared to the parent  Meripilus giganteus  protease. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 16/318,496filed Jan. 17, 2019, now allowed, which is a 35 U.S.C. 371 nationalapplication of international application no. PCT/EP2017/067883 filedJul. 14, 2017, which claims priority or the benefit under 35 U.S.C. 119of European application nos. 16180497.6 and 16195078.7, filed Jul. 21,2016 and Oct. 21, 2016, respectively, the contents of which are fullyincorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Production of fermentation products, such as ethanol, fromstarch-containing material is well-known in the art. Generally, twodifferent kinds of processes are used. The most commonly used process,often referred to as a “conventional process”, includes liquefyinggelatinized starch at high temperature using typically a bacterialalpha-amylase, followed by simultaneous saccharification andfermentation carried out in the presence of a glucoamylase and afermenting organism. Another well-known process, often referred to as a“raw starch hydrolysis”-process (RSH process) includes simultaneouslysaccharifying and fermenting granular starch below the initialgelatinization temperature typically in the presence of an acid fungalalpha-amylase and a glucoamylase.

U.S. Pat. No. 5,231,017-A discloses the use of an acid fungal proteaseduring ethanol fermentation in a process comprising liquefyinggelatinized starch with an alpha-amylase.

WO 2003/066826 discloses a raw starch hydrolysis process (RSH process)carried out on non-cooked mash in the presence of fungal glucoamylase,alpha-amylase and fungal protease.

WO 2007/145912 discloses a process for producing ethanol comprisingcontacting a slurry comprising granular starch obtained from plantmaterial with an alpha-amylase capable of solubilizing granular starchat a pH of 3.5 to 7.0 and at a temperature below the starchgelatinization temperature for a period of 5 minutes to 24 hours;obtaining a substrate comprising greater than 20% glucose, andfermenting the substrate in the presence of a fermenting organism andstarch hydrolyzing enzymes at a temperature between 10° C. and 40° C.for a period of 10 hours to 250 hours. Additional enzymes added duringthe contacting step may include protease.

WO 2014/037438 discloses serine proteases derived from Meripilusgiganteus, Trametes versicolor, and Dichomitus squalens and their use inanimal feed.

U.S. provisional application 62/232,903 discloses the use of theMeripilus giganteus S53 protease in the saccharification and/orfermentation step in a starch to ethanol process.

It is an object of the present invention to identify variants of the M.giganteus S53 proteases that will result in increased storage stability,in particular an increased thermo-stability of the variant proteasecompared to the wild type parent enzyme.

The present invention provides protease variants with improvedproperties compared to its parent.

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to a protease variantcomprising a modification at one or more position corresponding topositions 39, 50, 57, 60, 74, 81, 84, 109, 110, 111, 115, 117, 124, 128,142, 145, 146, 154, 182, 183, 187, 207, 209, 210, 212, 228, 267, 271,272, 274, 278, 280, 294, 317, 318, 320, 321, 322, 328, 343, 348, 362 or363 of the polypeptide of SEQ ID NO: 3, wherein the variant has proteaseactivity and wherein the variant has at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100% sequence identity to themature polypeptide of SEQ ID NO: 3, and wherein the variant hasincreased thermo-stability compared to the protease of SEQ ID NO: 3.

In a second aspect the present invention relates to protease variantcomprising a modification at position corresponding to position 39, 60,74, 81, 84, 109, 115, 117, 142, 145, 146, 154, 182, 183, 187, 209, 210,212, 228, 267, 272, 280, 294, 317, 318, 348 or 362 of the polypeptide ofSEQ ID NO: 3, wherein the variant has protease activity and wherein thevariant has at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3,wherein the protease is a serine protease belonging to the S53 familyand wherein the variant has increased residual activity compared to theprotease of SEQ ID NO: 3 and wherein the increased thermo-stability isincreased residual activity measured after incubation for 30 min at anelevated temperature in the range from 55 to 60 degrees Celsius.

In a third aspect the present invention relates to protease variantcomprising a modification at position corresponding to position 50, 57,60, 81, 84, 109, 110, 111, 124, 128, 142, 145, 146, 154, 182, 183, 207,209, 210, 228, 267, 271, 272, 274, 278, 280, 294, 317, 318, 320, 321,322, 328, 343, 362, or 363 of the polypeptide of SEQ ID NO: 3, whereinthe variant has protease activity and wherein the variant has at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%sequence identity to the polypeptide of SEQ ID NO: 3, wherein theprotease is a serine protease belonging to the S53 family and whereinthe protease is a serine protease belonging to the S53 family which hasan improved property relative to the parent wherein the improvedproperty is increased thermo-stability measured by TSA assay where Td isat least 59° C.

The present invention also relates to polynucleotides encoding thevariants; nucleic acid constructs, vectors, and host cells comprisingthe polynucleotides; and methods of producing the variants. In a furtheraspect the present invention relates to compositions comprising thevariants of the invention.

The present invention also relates to a process for producing afermentation product from starch-containing material comprisingsimultaneously saccharifying and fermenting starch-containing materialusing a carbohydrate-source generating enzymes and a fermenting organismat a temperature below the initial gelatinization temperature of saidstarch-containing material in the presence of a variant protease. Inanother aspect the present invention relates to a process for producinga fermentation product from starch-containing material comprising thesteps of: (a) liquefying starch-containing material in the presence ofan alpha-amylase; (b) saccharifying the liquefied material obtained instep (a) using a glucoamylse; (c)

fermenting using a fermenting organism; wherein a variant protease ofthe invention is present during step b) and/or c).

Definitions

Protease: The term “protease” (also designated peptidases, proteinases,peptide hydrolases, or proteolytic enzymes) means a proteolytic activity(EC 3.4) that catalyzes the cleavage of peptide bonds. For purposes ofthe present invention, serine protease activity is determined accordingto the procedure described in the Examples. In one aspect, the variantsof the present invention have at least 20%, e.g., at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or at least 100% of the protease activity of the mature polypeptideof SEQ ID NO: 2.

Protease activity: The term “protease activity” means proteolyticactivity (EC 3.4). There are several protease activity types such astrypsin-like proteases cleaving at the carboxyterminal side of Arg andLys residues and chymotrypsin-like proteases cleaving at thecarboxyterminal side of hydrophobic amino acid residues. Proteases ofthe invention are serine endopeptidases (EC 3.4.21) with acidicpH-optimum (pH optimum<pH 7).

Protease activity can be measured using any assay, in which a substrateis employed, that includes peptide bonds relevant for the specificity ofthe protease in question. Assay-pH and assay-temperature are likewise tobe adapted to the protease in question. Examples of assay-pH-values arepH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples of assay-temperaturesare 15, 20, 25, 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 95°C. Examples of general protease substrates are casein, bovine serumalbumin and haemoglobin. In the classical Anson and Mirsky method,denatured haemoglobin is used as substrate and after the assayincubation with the protease in question, the amount of trichloroaceticacid soluble haemoglobin is determined as a measurement of proteaseactivity (Anson, M. L. and Mirsky, A. E., 1932, J. Gen. Physiol. 16: 59and Anson, M. L., 1938, J. Gen. Physiol. 22: 79).

For the purpose of the present invention, protease activity wasdetermined using assays which are described in “Materials and Methods”,such as the Kinetic Suc-AAPF-pNA assay, Protazyme AK assay, KineticSuc-AAPX-pNA assay and o-Phthaldialdehyde (OPA). For the Protazyme AKassay, insoluble Protazyme AK (Azurine-Crosslinked Casein) substrateliberates a blue colour when incubated with the protease and the colouris determined as a measurement of protease activity. For theSuc-AAPF-pNA assay, the colourless Suc-AAPF-pNA substrate liberatesyellow paranitroaniline when incubated with the protease and the yellowcolour is determined as a measurement of protease activity.

Endo-protease/Exo-proteases: Polypeptides having protease activity, orproteases, are sometimes also designated peptidases, proteinases,peptide hydrolases, or proteolytic enzymes. Proteases may be of theexo-type (exopeptidases) that hydrolyse peptides starting at either endthereof, or of the endo-type that act internally in polypeptide chains(endopeptidases).

S53 protease: The term “S53” means a protease activity selected from:

(a) proteases belonging to the EC 3.4.21 enzyme group; and/or

(b) proteases belonging to the EC 3.4.14 enzyme group; and/or

(c) Serine proteases of the peptidase family S53 that comprises twodifferent types of peptidases: tripeptidyl aminopeptidases (exo-type)and endo-peptidases; as described in 1993, Biochem. J. 290:205-218 andin MEROPS protease database, release, 9.4 (31 Jan. 2011)(www.merops.ac.uk). The database is described in Rawlings, N. D.,Barrett, A. J. and Bateman, A., 2010, “MEROPS: the peptidase database”,Nucl. Acids Res. 38: D227-D233.

For determining whether a given protease is a Serine protease, and afamily S53 protease, reference is made to the above Handbook and theprinciples indicated therein. Such determination can be carried out forall types of proteases, be it naturally occurring or wild-typeproteases; or genetically engineered or synthetic proteases.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has proteaseactivity.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parent. Suchimproved properties include, but are not limited to, increased stabilityunder storage conditions, increased thermo-stability, and increasedresidual activity.

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 199 to 564 of SEQ ID NO: 2. Amino acids 1 to17 of SEQ ID NO: 2 are a signal peptide, and amino acids 18 to 198 are apropeptide. The N-terminals of the mature S53 polypeptides usedaccording to the present invention were experimentally confirmed basedon EDMAN N-terminal sequencing data and Intact MS data. The maturepolypeptides are also included as SEQ ID NO: 3 (mature S53 protease 3from Meripilus giganteus. It is known in the art that a host cell mayproduce a mixture of two of more different mature polypeptides (i.e.,with a different C-terminal and/or N-terminal amino acid) expressed bythe same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving protease activity. In one aspect, the mature polypeptide codingsequence is nucleotides 595 to 1692 of SEQ ID NO: 1. Nucleotides 1 to 51of SEQ ID NO: 1 encode a signal peptide, nucleotides 52 to 594 encode apropeptide.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Modification: The term “modification(s)” is in the context of thepresent invention to be understood as a substitution, insertion, and/ordeletion, at one or more (e.g., several) positions. A substitution meansreplacement of the amino acid occupying a position with a differentamino acid; a deletion means removal of the amino acid occupying aposition; and an insertion means adding an amino acid adjacent to andimmediately following the amino acid occupying a position.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences. In one embodiment the one or more control sequences areheterologous (of different origin/species) to the coding sequenceencoding the polypeptide of the invention.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent protease: The term “parent” or “parent protease” meansany polypeptide with protease activity to which an alteration is made toproduce the enzyme variants of the present invention.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having protease activity. In one aspect, a subsequence containsat least 1098 nucleotides (e.g., nucleotides 595 to 1692 of SEQ ID NO:1).

Variant: The term “variant” means a polypeptide having protease activitycomprising a modification(s) at one or more (e.g., several) positions.The variants of the present invention have at least 20%, e.g., at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, or at least 100% of the protease activity of themature polypeptide of SEQ ID NO: 2, disclosed herein as SEQ ID NO: 3.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Wild-type protease: The term “wild-type” protease means a proteaseexpressed by a naturally occurring microorganism, such as a bacterium,yeast, or filamentous fungus found in nature.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide comprisedin SEQ ID NO: 2 is used to determine the corresponding amino acidresidue in another protease. The amino acid sequence of another proteaseis aligned with the mature polypeptide comprised in SEQ ID NO: 2(disclosed herein as SEQ ID NO: 3), and based on the alignment, theamino acid position number corresponding to any amino acid residue inthe mature polypeptide comprised in SEQ ID NO: 2 is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 orlater. The parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix.

Identification of the corresponding amino acid residue in anotherprotease than the Meripilus giganteus S53 protease can be determined byan alignment of multiple polypeptide sequences using several computerprograms including, but not limited to, MUSCLE (multiple sequencecomparison by log-expectation; version 3.5 or later; Edgar, 2004,Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or later;Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh etal., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh, 2007,Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in MolecularBiology 537: 39-64; Katoh and Toh, 2010, Bioinformatics 26: 1899-1900),and EMBOSS EMMA employing ClustalW (1.83 or later; Thompson et al.,1994, Nucleic Acids Research 22: 4673-4680), using their respectivedefault parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 2 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine at position 226 withalanine is designated as “Thr226Ala” or “T226A”. Multiple mutations areseparated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple modification. Variants comprising multiple modifications areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.

Different modifications. Where different modification can be introducedat a position, the different modifications are separated by a comma,e.g., “Arg170Tyr,Glu” represents a substitution of arginine at position170 with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala”designates the following variants: “Tyr167Gly+Arg170Gly”,“Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to protease variants, comprising amodification(s) at one or more (e.g., several) positions correspondingto specific positions of the mature polypeptide disclosed as SEQ ID NO:3 (a parent protease), wherein the variant has protease activity. Asexplained herein the specific position numbers may change in case themature parent protease is different from SEQ ID NO: 3. The improvedproperties of the variants of the invention falls in the followingcategories, e.g., increased stability, e.g., increased thermostability(measured as increase in thermal denaturation temperature, Td, and/orincreased residual activity by the Suc-AAPF assay after incubation for30 min at an elevated temperature in the range from 55 to 60 degreesCelsius as described in detail in the examples).

Variants

The present invention provides a protease variant comprising amodification at one or more position corresponding to positions 39, 50,57, 60, 74, 81, 84, 109, 110, 111, 115, 117, 124, 128, 142, 145, 146,154, 182, 183, 187, 207, 209, 210, 212, 228, 267, 271, 272, 274, 278,280, 294, 317, 318, 320, 321, 322, 328, 343, 348, 362 or 363 of thepolypeptide of SEQ ID NO: 3, wherein each modification is independentlya substitution, insertion or deletion. In one embodiment, themodification is a substitution. In another embodiment, the modificationis a deletion.

The present invention provides a protease variant comprising amodification at one or more position corresponding to positions 39, 50,57, 60, 74, 81, 84, 109, 110, 111, 115, 117, 124, 128, 142, 145, 146,154, 182, 183, 187, 207, 209, 210, 212, 228, 267, 271, 272, 274, 278,280, 294, 317, 318, 320, 321, 322, 328, 343, 348, 362 or 363 of thepolypeptide of SEQ ID NO: 3, wherein the variant has protease activityand wherein the variant has at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the maturepolypeptide of SEQ ID NO: 3, and wherein the variant has increasedthermo-stability compared to the protease of SEQ ID NO: 3.

In an embodiment, the variant has sequence identity of at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100%, to the amino acid sequence of the matureparent protease.

In another embodiment, the variant has at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, such as at least 96%, at least 97%, at least 98%, or at least99%, but less than 100%, sequence identity to the polypeptide of SEQ IDNO: 3.

In one aspect, the number of substitutions in the variants of thepresent invention is 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 substitutions.

In one specific aspect the invention relates to a protease variantcomprising a modification at position corresponding to positions 39, 60,74, 81, 84, 109, 115, 142, 145, 146, 154, 182, 183, 187, 209, 210, 212,228, 267, 272, 280, 294, 317, 318, 348 or 362 of the polypeptide of SEQID NO: 3, wherein the variant has protease activity and wherein thevariant has at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3,wherein the protease is a serine protease belonging to the S53 familyand wherein the variant has increased thermo-stability compared to theprotease of SEQ ID NO: 3.

More specifically the variant comprises a modification which is asubstitution at position corresponding to positions 39, 50, 57, 60, 74,81, 84, 109, 110, 111, 115, 117, 124, 128, 142, 145, 146, 154, 182, 183,187, 207, 209, 212, 228, 267, 271, 272, 274, 278, 280, 294, 317, 318,320, 321, 322, 328, 343, 348, 362 or 363 of the polypeptide of SEQ IDNO: 3, wherein the variant has protease activity and wherein the varianthas at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100% sequence identity to the polypeptide of SEQ ID NO: 3, whereinthe protease is a serine protease belonging to the S53 family andwherein the variant has increased thermo-stability compared to theprotease of SEQ ID NO: 3.

More specifically the variant comprises a modification which is adeletion at position corresponding to position 318 or 210 of thepolypeptide of SEQ ID NO: 3, wherein the variant has protease activityand wherein the variant has at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3, wherein the protease is a serine protease belonging to theS53 family and wherein the variant has increased thermo-stabilitycompared to the protease of SEQ ID NO: 3.

In a further specific embodiment the variant comprises or consists of atleast one substitutions and/or deletions selected from the groupconsisting of: I39M, I39R, I39L, I39C, S500, K57R, 560P, 560D, E74W,E81A, E81E, E81K, E81R, I84C, D109N, D109P, D110N, F111P, N115D, N115L,E117D, N124Q, N124L, N124W, G128A, Q142R, Q142W, N145A, N145D, N145E,N145G, N145K, N145Q, N145V, T146A, T146D, T146E, T146W, T146Y, Q154R,Q154V, Q154W, Q154Y, Q182G, Q182R, S183L, S183P, S187L, Q207R, V209L,E212E, 1228R, D267N, V271C, S272C, S272R, S272V, G274G, G278S, D280N,S294A, S317A, S317G, S317S, S318N, G320C, K321A, K321G, A322S, T328C,K343C, P348A, T362A, A363C, S318* and S210* of the polypeptide of SEQ IDNO: 3, wherein the variant has protease activity and wherein the varianthas at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%, but lessthan 100% sequence identity to the polypeptide of SEQ ID NO: 3, whereinthe protease is a serine protease belonging to the S53 family andwherein the variant has increased thermo-stability compared to theprotease of SEQ ID NO: 3.

In another aspect, the protease variant comprises a modification atposition corresponding to position 39, 60, 74, 81, 84, 109, 115, 117,142, 145, 146, 154, 182, 183, 187, 209, 210, 212, 228, 267, 272, 280,294, 317, 318, 348 or 362 of the polypeptide of SEQ ID NO: 3, whereinthe variant has protease activity and wherein the variant has at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%sequence identity to the polypeptide of SEQ ID NO: 3, wherein theprotease is a serine protease belonging to the S53 family, and whereinthe increased thermo-stability is increased residual activity measuredafter incubation for 30 min at an elevated temperature in the range from55 to 60 degrees Celsius.

More specifically the variant comprises a modification which is adeletion at position corresponding to position 318 or 210 of thepolypeptide of SEQ ID NO: 3 wherein the variant has protease activityand wherein the variant has at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3, wherein the protease is a serine protease belonging to theS53 family, and wherein the increased thermo-stability is increasedresidual activity measured after incubation for 30 min at an elevatedtemperature in the range from 55 to 60 degrees Celsius.

More specifically the variant comprises a modification which is asubstitution at position corresponding to positions 39, 60, 74 81, 84,109, 115, 142, 145, 146, 154, 182, 183, 187, 209, 212, 228, 267, 272,280, 294, 317, 348 or 362 of the polypeptide of SEQ ID NO: 3, whereinthe variant has protease activity and wherein the variant has at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%sequence identity to the polypeptide of SEQ ID NO: 3, wherein theprotease is a serine protease belonging to the S53 family, wherein theincreased thermo-stability is increased residual activity measured afterincubation for 30 min at a temperature in the range from 55 to 60degrees Celsius.

In a further specific embodiment the variant comprises or consists ofone or more substitutions and/or deletions selected from the groupconsisting of: I39M, I39R, I39L, I39C, S60D, I84C N115D, N115L, E117D,N145G, N145Q, N145V, N145D, N145K, N145K, N145A, N145E, S183L, S183P,D280N, Q182G, Q182R, E81R, E81K, E81E, E81A, I84C, Q154V, Q142W, Q142R,T146A, T146W, T146Y, T146E, T146D, 1228R, D267N, S272V, S272R, E212E,S294A, T362A, E74W, S187L, P348A, D109P, S317A, S317G, S317S, S317A,S318* and S210* of the polypeptide of SEQ ID NO: 3, wherein the varianthas protease activity and wherein the variant has at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%, but less than 100% sequence identityto the polypeptide of SEQ ID NO: 3, and wherein the increasedthermo-stability is increased residual activity measured afterincubation for 30 min at a temperature in the range from 55 to 60degrees Celsius.

In a further specific embodiment the variant comprises at least one ofthe following modifications or combination of modifications:

-   -   N115L;    -   S183P;    -   D280N;    -   N115D;    -   N115L+Q182G;    -   N115L+Q182R;    -   E81R+S183P;    -   E81K+S183P;    -   S183P+Q154V;    -   S183P+Q142W;    -   Q142R+S183P;    -   S183P+T146A;    -   S183P+T146W;    -   S183P+I228R;    -   S183P+D267N;    -   S183P+S272V;    -   S183P+S272R;    -   T146W+D280N;    -   T146Y+S183P;    -   S183P+E212E;    -   S183P+S294A;    -   S183P+T362A;    -   S183P+S294A;    -   S183P+E74W;    -   S183P+E81E;    -   S183P+E81A;    -   N115L+S183L+S187L;    -   S183L+V209L+S210*;    -   D109P+V209L+S210*;    -   N115D+V209L+S210*;    -   E81R+V209L+S210*;    -   D109P+V209L+S210*;    -   N115D+V209L+S210*;    -   E81R+V209L+S210*;    -   T146W+S183P+D280N;    -   I84C+S183P+S272C;    -   I39M+Q142R+S183P;    -   I39R+Q142R+S183P;    -   I39L+Q142R+S183P;    -   I39C+Q142R+S183P;    -   E117D+Q142R+S183P;    -   S60D+Q142R+S183P;    -   N115L+S183L+S187L+P348A;    -   D109P+S183P+V209L+S210*;    -   N115D+S183P+V209L+S210*;    -   E81R+S183P+V209L+S210*;    -   V209L+S210*+S317A+S318*;    -   Q142R+N145G+T146E+S183P;    -   Q142R+N145Q+T146D+S183P;    -   Q142R+N145V+T146E+S183P;    -   Q142R+N145D+T146E+S183P;    -   Q142R+N145K+T146E+S183P;    -   Q142R+N145A+T146D+S183P;    -   Q142R+N145E+T146E+S183P;    -   N115L+S183L+S187L+V209W+S210*;    -   N115L+S183L+S187L+V209L+S210*;    -   N115L+S183L+S187L+S317G+S318*;    -   N115L+S183L+S187L+S317S+S318*;    -   N115L+S183L+S187L+S317A+S318*;    -   E81R+V209L+S210*+S317A+S318*; and wherein the increased        thermo-stability is increased residual activity measured after        incubation for 30 min at a temperature in the range from 55 to        60 degrees Celsius. More particularly the variants have a        residual activity of at least 10%, particularly at least 12%,        more particularly at least 15%, measured after incubation for 30        minutes at 56° C.

In a further specific embodiment the variant comprises at least one ofthe following modifications or combination of modifications:

-   -   N115L+Q182G;    -   N115D;    -   Q142R+S183P;    -   Q142R+N145G+T146E+S183P;    -   Q142R+N145Q+T146D+S183P;    -   Q142R+N145V+T146E+S183P;    -   Q142R+N145D+T146E+S183P;    -   Q142R+N145K+T146E+S183P;    -   Q142R+N145A+T146D+S183P;    -   I39M+Q142R+S183P;    -   Q142R+N145E+T146E+S183P;    -   I39R+Q142R+S183P;    -   I39L+Q142R+S183P;    -   E117D+Q142R+S183P;    -   S60D+Q142R+S183P; and wherein the variant has residual activity        of at least 30% measured after incubation for 30 min at an        elevated temperature of 57 degrees Celsius.

In a further specific embodiment the variant comprises at least one ofthe following modifications or combination of modifications:

-   -   Q142R+S183P;    -   Q142R+N145G+T146E+S183P;    -   Q142R+N145Q+T146D+S183P;    -   Q142R+N145V+T146E+S183P;    -   Q142R+N145D+T146E+S183P;    -   Q142R+N145K+T146E+S183P;    -   Q142R+N145A+T146D+S183P;    -   I39M+Q142R+S183P;    -   Q142R+N145E+T146E+S183P;    -   I39R+Q142R+S183P;    -   I39L+Q142R+S183P;    -   E117D+Q142R S183P;    -   Q142R+S183P;    -   S60D+Q142R+S183P;    -   Q142R+S183P; and wherein the variant has residual activity of at        least 70% measured after incubation for 30 min at 57 degrees        Celsius.    -   In a further specific embodiment the variant comprises at least        one of the following modification or combination of        modifications:    -   Q142R+S183P;    -   I39C+Q142R+S183P;    -   E117D+Q142R+S183P;    -   Q142R+S183P;    -   S60D+Q142R+S183P; and wherein the variant has residual activity        of at least 40% measured after incubation for 30 min at 60        degrees Celsius.

In a further specific embodiment the variant comprises at least one ofthe following modification or combination of modifications:

-   -   Q142R+S183P;    -   I39C+Q142R+S183P; and wherein the variant has residual activity        of at least 40% measured after incubation for 30 minutes at 62°        C.

The present invention relates to a variant comprising a modification atposition corresponding to position 50, 57, 60, 81, 84, 109, 110, 111,124, 128, 142, 145, 146, 154, 182, 183, 207, 209, 210, 228, 267, 271,272, 274, 278, 280, 294, 317, 318, 320, 321, 322, 328, 343, 362, or 363of the polypeptide of SEQ ID NO: 3, wherein the variant has proteaseactivity and wherein the variant has at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100% sequence identity to thepolypeptide of SEQ ID NO: 3, wherein the protease is a serine proteasebelonging to the S53 family.

More specifically the variant comprises a modification which is asubstitution at position corresponding to positions 50, 57, 60, 81, 84,109, 110, 111, 124, 128, 142, 145, 146, 154, 182, 183, 207, 209, 228,267, 271, 272, 274, 278, 280, 294, 317, 318, 320, 321, 322, 328, 343,362, or 363 of the polypeptide of SEQ ID NO: 3, wherein the variant hasprotease activity and wherein the variant has at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%, but less than 100% sequence identityto the polypeptide of SEQ ID NO: 3, wherein the protease is a serineprotease belonging to the S53 family; and wherein the increase inthermo-stability is an increase in thermal denaturation temperaturemeasured by TSA. In particular, the increased thermo-stability measuredas Td by TSA assay is at least 59° C.

More specifically the variant comprises a modification which is adeletion at position corresponding to position 318 or 210 of thepolypeptide of SEQ ID NO: 3, wherein the variant has protease activityand wherein the variant has at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3, wherein the protease is a serine protease belonging to theS53 family; and wherein the increase in thermo-stability is an increasein thermal denaturation temperature measured by TSA. In particular, theincreased thermo-stability measured as Td by TSA assay is at least 59°C.

In a further specific embodiment the variant comprises or consists ofone or more substitution(s) and/or deletion(s) selected from the groupconsisting of S50C, K57R, S60P, E81R, I84C, D109P, D109N, D110N, F111P,N124L, N124W, N124Q, G128A, Q142R, Q142W, N145V, N145D, N145A, T146A,T146W, T146E, T146D, Q154V, Q154W, Q154,R, Q154Y, Q182G, Q182R, S183P,S183L, Q207R, V209L, 1228R, D267N, V271C, S272V, S272C, S272R, G274G,G278S, D280N, S294A, S317A, S318N, G320C, K321G, K321A, A322S, T328C,K343C, T362A, A363C, S318* and S210* of the polypeptide of SEQ ID NO: 3,wherein the variant has protease activity and wherein the variant has atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe increased thermo-stability measured as Td by TSA assay is at least59° C.

In a further specific embodiment, the variant comprises at least one ofthe following modifications or combination of modifications:

-   -   S183P;    -   D280N;    -   K57R+S183P;    -   D109P+S183P+V209L+S210*;    -   E81R+S183P+V209L+S210*;    -   E81R+V209L+S210*;    -   Q154V+S183P;    -   Q142W+S183P;    -   Q142R+S183P;    -   T146A+S183P;    -   T146W+S183P;    -   S183P+I228R;    -   S183P+D267N;    -   S183P+S272V;    -   E81R+V209L+S210*+S317A+S318*;    -   S183P+T328C+K343C;    -   S183P+G3200+A363C;    -   T146W+D280N;    -   T146W+S183L D+280N;    -   T146W;    -   T146W+S183P+D280N;    -   T146Y+S183P;    -   S183P+Q207R;    -   S500+S183P+V271C;    -   I84C+S183P+S272C;    -   Q142W+T146W+S183P;    -   Q142W+T146W+S183P+D280N;    -   S183P+S294A;    -   S183P+K321G;    -   S183P+T362A;    -   Q182G;    -   Q142W+T146W+Q182R;    -   S272V;    -   S272R;    -   S60P;    -   D109N+D110N;    -   F111P;    -   G128A;    -   G278S;    -   S318N+K321A+A322S;    -   E81R+T146W;    -   E81R+Q142R+S183P;    -   E81R+Q142W+S183P    -   S183P+G274G;    -   E81R;    -   N124L+Q142R+S183P;    -   N124W+Q142R+S183P;    -   N124Q+Q142R+S183P;    -   Q142R+N145V+T146E+S183P;    -   Q142R+N145D+T146E+S183P;    -   Q142R+N145A+T146D+S183P; and wherein the increased        thermo-stability measured as Td by TSA assay is at least 59° C.

In a further specific embodiment, the variant comprises at least one ofthe following modifications or combination of modifications:

-   -   D280N;    -   D109P+S183P+V209L+S210*;    -   E81R+S183P+V209L+S210*;    -   E81R+V209L+S210*;    -   Q154V+S183P;    -   Q142W+S183P;    -   Q142R+S183P;    -   T146A+S183P;    -   T146W+S183P;    -   S183P+D267N;    -   S183P+S272V;    -   E81R+V209L+S210*+S317A+S318*;    -   T146W+D280N;    -   T146W+S183L+D280N;    -   T146W;    -   T146W+S183P+D280N;    -   T146Y+S183P;    -   S183P+Q207R;    -   S50C+S183P+V271C;    -   I84C+S183P+S272C;    -   Q142W+T146W+S183P;    -   Q142W+T146W+S183P+D280N;    -   S183P+S294A;    -   Q142W+T146W+Q182R;    -   S272V;    -   S272R;    -   S60P;    -   E81R+T146W;    -   E81R+Q142R+S183P;    -   E81R+Q142W+S183P;    -   S183P+G274G;    -   E81R;    -   N124L+Q142R+S183P;    -   N124W+Q142R+S183P;    -   N124Q+Q142R+S183P;    -   Q142R+N145V+T146E+S183P;    -   Q142R+N145D+T146E+S183P;    -   Q142R+N145A+T146D+S183P; and wherein the increased        thermo-stability measured as Td by TSA assay is at least 61° C.

In a further specific embodiment, the variant comprises at least one ofthe following modifications or combination of modifications:

-   -   E81R+S183P+V209L+S210*;    -   Q142R+S183P;    -   T146W+D280N;    -   T146W+S183L+D280N;    -   T146W+S183P+D280N;    -   S500+S183P+V271C;    -   I84C+S183P+S272C;    -   Q142W+T146W+S183P+D280N;    -   S272V;    -   E81R+T146W;    -   E81R+Q142R+S183P;    -   N124L+Q142R+S183P;    -   N124W+Q142R+S183P;    -   N124Q+Q142R+S183P;    -   Q142R+N145V+T146E+S183P;    -   Q142R+N145D+T146E+S183P;    -   Q142R+N145A+T146D+S183P; and wherein the increased        thermo-stability measured as Td by TSA assay is at least 63° C.

In a further specific embodiment, the variant comprises at least one ofthe following modifications or combination of modifications:

-   -   Q142R+S183P;    -   S50C+S183P+V271C;    -   E81R+Q142R+S183P;    -   N124L+Q142R+S183P;    -   N124Q+Q142R+S183P;    -   Q142R+N145V+T146E+S183P;    -   Q142R+N145D+T146E+S183P; or    -   Q142R+N145A+T146D+S183P; and wherein the increased        thermo-stability measured as Td by TSA assay is at least 65° C.

The variants may further comprise one or more additional modification(s)at one or more (e.g., several) other positions. Such furthermodifications may preferably not change the properties of the proteasevariants of the present invention.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Therefore even though the protease variants according to the inventionmay only comprise one specific substitution providing the improvedproperty according to the invention it may still have additionmodifications leading to a variant protease having at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity, to the amino acidsequence of the mature parent protease, e.g., the protease of SEQ ID NO:3. These additional modification should preferably not significantlychange the improved properties of the variant protease.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for protease activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64.

In an embodiment, the variant has improved (increased) thermo-stabilitycompared to the parent enzyme, e.g., the polypeptide of SEQ ID NO: 3.

In an embodiment, the variant has improved (increased) residual activitycompared to parent enzyme, e.g., the polypeptide of SEQ ID NO: 3.

In an embodiment, the variant has improved (increased) thermal meltingtemperature compared to parent enzyme, e.g., the polypeptide of SEQ IDNO: 3.

Parent Proteases

The parent protease may be (a) a polypeptide having at least 80%sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) apolypeptide encoded by a polynucleotide that hybridizes under highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) the full-lengthcomplement of (i) or (ii); or (c) a polypeptide encoded by apolynucleotide having at least 80% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1.

In an aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 2 of at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%, which haveprotease activity. In one aspect, the amino acid sequence of the parentdiffers by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,from the mature polypeptide of SEQ ID NO: 2.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 3. In another aspect, the parent comprises orconsists of amino acids 199 to 564 of SEQ ID NO: 2.

In another aspect, the parent is encoded by a polynucleotide thathybridizes under high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence thereof, or (iii) the full-length complementof (i) or (ii) (Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used todesign nucleic acid probes to identify and clone DNA encoding a parentfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of a cell of interest,following standard Southern blotting procedures, in order to identifyand isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1 or a subsequence thereof, the carrier material is usedin a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iii) the cDNA sequence thereof; (iv) the full-lengthcomplement thereof; or (v) a subsequence thereof; under high to veryhigh stringency conditions. Molecules to which the nucleic acid probehybridizes under these conditions can be detected using, for example,X-ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1. In another aspect, the nucleic acid probe isnucleotides 595 to 1692 of SEQ ID NO: 1. In another aspect, the nucleicacid probe is a polynucleotide that encodes the polypeptide of SEQ IDNO: 2; the mature polypeptide thereof; or a fragment thereof. In anotheraspect, the nucleic acid probe is SEQ ID NO: 1 or the cDNA sequencethereof.

In another embodiment, the parent is encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

In another aspect, the parent is a Meripilus giganteus S53 protease,e.g., the protease of SEQ ID NO: 2 or the mature polypeptide thereof,disclosed herein as SEQ ID NO: 3.

Preparation of Variants

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to polynucleotides encoding a variantof the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the variant. Any leader that is functional in the host cell maybe used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Myceliophthorathermophila laccase (WO 95/33836), Rhizomucor miehei asparticproteinase.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are Aspergillus nidulans orAspergillus oryzae niaD, niiA, amdS and pyrG genes and a Streptomyceshygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. In a particular embodiment, therecombinant host cell comprises the polynucleotide encoding a trehalasepolypeptide of the present invention, wherein the said polynucleotide isheterologous (of different origin/species) to the host cell. A constructor vector comprising a polynucleotide is introduced into a host cell sothat the construct or vector is maintained as a chromosomal integrant oras a self-replicating extra-chromosomal vector as described earlier. Theterm “host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the variant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a recombinant host cell of the presentinvention under conditions suitable for expression of the variant; and(b) recovering the variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants. These detection methods include, but are notlimited to, use of specific antibodies, formation of an enzyme product,or disappearance of an enzyme substrate. For example, an enzyme assaymay be used to determine the activity of the variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Enzyme Compositions

The present invention also relates to compositions comprising variantprotease of the invention. Preferably, the compositions are enriched insuch a protease. The term “enriched” indicates that the pullulanaseactivity of the composition has been increased, e.g., with an enrichmentfactor of at least 1.1.

The compositions may comprise the variant S53 protease as the majorenzymatic component, e.g., a mono-component composition. Alternatively,the compositions may comprise multiple enzymatic activities, such as thevariant S53 protease and one or more (e.g., several) enzymes selectedfrom the group consisting of hydrolase, isomerase, ligase, lyase,oxidoreductase, or transferase, e.g., an alpha-galactosidase,alpha-glucosidase, aminopeptidase, alpha-amylase, beta-amylase,beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, glucoamylase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, protease, ribonuclease, transglutaminase, orxylanase. In one embodiment the composition comprises a variant S53protease of the invention and a carbohydrate-source generating enzymeand optionally an alpha-amylase. In one particular embodiment thecomposition comprises a variant S53 protease and a glucoamylase.Preferably the enzyme activities comprised in the composition areselected from the variant S53 protease of the invention and one or moreenzymes selected from the group consisting of glucoamylase, fungalalpha-amylase.

In an embodiment the glucoamylase comprised in the composition is offungal origin, preferably from a stain of Aspergillus, preferably A.niger, A. awamori, or A. oryzae; or a strain of Trichoderma, preferablyT. reesei; or a strain of Talaromyces, preferably T. emersonii or astrain of Trametes, preferably T. cingulata, or a strain of Pycnoporus,preferable P. sanguineus, or a strain of Gloeophyllum, such as G.serpiarium or G. trabeum, or a strain of the Nigrofomes.

In an embodiment the glucoamylase is derived from Trametes, such as astrain of Trametes cingulata, such as the one shown in SEQ ID NO: 4herein.

In an embodiment the glucoamylase is selected from the group consistingof:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 4 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the polypeptide of SEQ ID NO: 4 herein.

In an embodiment the glucoamylase is derived from Talaromyces, such as astrain of Talaromyces emersonii, such as the one shown in SEQ ID NO: 5herein.

In an embodiment the glucoamylase is selected from the group consistingof:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 5 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the polypeptide of SEQ ID NO: 5 herein.

In an embodiment the glucoamylase is derived from a strain of the genusPycnoporus, in particular a strain of Pycnoporus sanguineus described inWO 2011/066576 (SEQ ID NOs 2, 4 or 6), such as the one shown as SEQ IDNO: 4 in WO 2011/066576.

In an embodiment the glucoamylase is selected from the group consistingof:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 6 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at SEQ ID NO: 6 herein.

In an embodiment the glucoamylase is derived from a strain of the genusGloeophyllum, such as a strain of Gloeophyllum sepiarium or Gloeophyllumtrabeum, in particular a strain of Gloeophyllum as described in WO2011/068803 (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16). In a preferredembodiment the glucoamylase is the Gloeophyllum sepiarium shown in SEQID NO:

2 in WO 2011/068803 or SEQ ID NO: 7 herein.

In an embodiment the glucoamylase is derived from Gloeophyllumserpiarium, such as the one shown in SEQ ID NO: 7 herein. In anembodiment the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 7 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the polypeptide of SEQ ID NO: 7 herein.

In another embodiment the glucoamylase is derived from Gloeophyllumtrabeum such as the one shown in SEQ ID NO: 8 herein. In an embodimentthe glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 8 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the polypeptide of SEQ ID NO: 8 herein.

In an embodiment the glucoamylase is derived from a strain of the genusNigrofomes, in particular a strain of Nigrofomes sp. disclosed in WO2012/064351.

Glucoamylases may in an embodiment be added to the saccharificationand/or fermentation in an amount of 0.0001-20 AGU/g DS, preferably0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, such as 0.1-2AGU/g DS.

Commercially available compositions comprising glucoamylase include AMG200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME™ EXCEL and AMG™ E(from Novozymes A/S); OPTIDEX™ 300, GC480, GC417 (from DuPont.);AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™ and G990 ZR(from DuPont).

In addition to a glucoamylase the composition may further comprise analpha-amylase. Particularly the alpha-amylase is an acid fungalalpha-amylase. A fungal acid stable alpha-amylase is an alpha-amylasethat has activity in the pH range of 3.0 to 7.0 and preferably in the pHrange from 3.5 to 6.5, including activity at a pH of about 4.0, 4.5,5.0, 5.5, and 6.0.

Preferably the acid fungal alpha-amylase is derived from the genusAspergillus, especially a strain of A. terreus, A. niger, A. oryzae, A.awamori, or Aspergillus kawachii, or from the genus Rhizomucor,preferably a strain the Rhizomucor pusillus, or the genus Meripilus,preferably a strain of Meripilus giganteus.

In a preferred embodiment the alpha-amylase is derived from a strain ofthe genus Rhizomucor, preferably a strain the Rhizomucor pusillus, suchas one shown in SEQ ID NO: 3 in WO 2013/006756, such as a Rhizomucorpusillus alpha-amylase hybrid having an Aspergillus niger linker andstarch-binding domain, such as the one shown in SEQ ID NO: 9 herein, ora variant thereof.

In an embodiment the alpha-amylase is selected from the group consistingof:

(i) an alpha-amylase comprising the polypeptide of SEQ ID NO: 9 herein;

(ii) an alpha-amylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the polypeptide of SEQ ID NO: 9 herein.

In a preferred embodiment the alpha-amylase is a variant of thealpha-amylase shown in SEQ ID NO: 9 having at least one of the followingsubstitutions or combinations of substitutions: D165M; Y141W; Y141R;K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W;G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R;Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N;Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C;Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C;G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C (using SEQ IDNO: 9 for numbering).

In an embodiment the alpha-amylase is derived from a Rhizomucor pusilluswith an Aspergillus niger glucoamylase linker and starch-binding domain(SBD), preferably disclosed as SEQ ID NO: 9 herein, preferably havingone or more of the following substitutions: G128D, D143N, preferablyG128D+D143N (using SEQ ID NO: 9 for numbering), and wherein thealpha-amylase variant has at least 75% identity preferably at least 80%,more preferably at least 85%, more preferably at least 90%, morepreferably at least 91%, more preferably at least 92%, even morepreferably at least 93%, most preferably at least 94%, and even mostpreferably at least 95%, such as even at least 96%, at least 97%, atleast 98%, at least 99%, but less than 100% identity to the polypeptideof SEQ ID NO: 9 herein.

In a preferred embodiment the ratio between glucoamylase andalpha-amylase present and/or added during saccharification and/orfermentation may preferably be in the range from 500:1 to 1:1, such asfrom 250:1 to 1:1, such as from 100:1 to 1:1, such as from 100:2 to100:50, such as from 100:3 to 100:70.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Forinstance, the composition may be in the form of granulate ormicrogranulate. The variant may be stabilized in accordance with methodsknown in the art.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

The enzyme composition of the present invention may be in any formsuitable for use, such as, for example, a crude fermentation broth withor without cells removed, a cell lysate with or without cellular debris,a semi-purified or purified enzyme composition, or a host cell, as asource of the enzymes.

The enzyme composition may be a dry powder or granulate, a non-dustinggranulate, a liquid, a stabilized liquid, or a stabilized protectedenzyme. Liquid enzyme compositions may, for instance, be stabilized byadding stabilizers such as a sugar, a sugar alcohol or another polyol,and/or lactic acid or another organic acid according to establishedprocesses.

Use of the Variant Proteases of the Invention

Starch Processing

Native starch consists of microscopic granules, which are insoluble inwater at room temperature. When aqueous starch slurry is heated, thegranules swell and eventually burst, dispersing the starch moleculesinto the solution. At temperatures up to about 50° C. to 75° C. theswelling may be reversible. However, with higher temperatures anirreversible swelling called “gelatinization” begins. During this“gelatinization” process there is a dramatic increase in viscosity.Granular starch to be processed may be a highly refined starch quality,preferably at least 90%, at least 95%, at least 97% or at least 99.5%pure or it may be a more crude starch-containing materials comprising(e.g., milled) whole grains including non-starch fractions such as germresidues and fibers. The raw material, such as whole grains, may bereduced in particle size, e.g., by milling, in order to open up thestructure and allowing for further processing. In dry milling wholekernels are milled and used. Wet milling gives a good separation of germand meal (starch granules and protein) and is often applied at locationswhere the starch hydrolysate is used in the production of, e.g., syrups.Both dry and wet milling is well known in the art of starch processingand may be used in a process of the invention. Methods for reducing theparticle size of the starch containing material are well known to thoseskilled in the art.

As the solids level is 30-40% in a typical industrial process, thestarch has to be thinned or “liquefied” so that it can be suitablyprocessed. This reduction in viscosity is primarily attained byenzymatic degradation in current commercial practice.

Liquefaction is carried out in the presence of an alpha-amylase,preferably a bacterial alpha-amylase and/or acid fungal alpha-amylase.In an embodiment, a phytase is also present during liquefaction. In anembodiment, viscosity reducing enzymes such as a xylanase and/orbeta-glucanase is also present during liquefaction.

During liquefaction, the long-chained starch is degraded into branchedand linear shorter units (maltodextrins) by an alpha-amylase.Liquefaction may be carried out as a three-step hot slurry process. Theslurry is heated to between 60-95° C. (e.g., 70-90° C., such as 77-86°C., 80-85° C., 83-85° C.) and an alpha-amylase is added to initiateliquefaction (thinning).

The slurry may in an embodiment be jet-cooked at between 95-140° C.,e.g., 105-125° C., for about 1-15 minutes, e.g., about 3-10 minutes,especially around 5 minutes. The slurry is then cooled to 60-95° C. andmore alpha-amylase is added to obtain final hydrolysis (secondaryliquefaction). The jet-cooking process is carried out at pH 4.5-6.5,typically at a pH between 5 and 6. The alpha-amylase may be added as asingle dose, e.g., before jet cooking.

The liquefaction process is carried out at between 70-95° C., such as80-90° C., such as around 85° C., for about 10 minutes to 5 hours,typically for 1-2 hours. The pH is between 4 and 7, such as between 4.5and 5.5. In order to ensure optimal enzyme stability under theseconditions, calcium may optionally be added (to provide 1-60 ppm freecalcium ions, such as about 40 ppm free calcium ions). After suchtreatment, the liquefied starch will typically have a “dextroseequivalent” (DE) of 10-15.

Generally liquefaction and liquefaction conditions are well known in theart.

Alpha-amylases for use in liquefaction are preferably bacterial acidstable alpha-amylases. Particularly the alpha-amylase is from anExiguobacterium sp. or a Bacillus sp. such as e.g., Bacillusstearothermophilus or Bacillus licheniformis.

In an embodiment the alpha-amylase is from the genus Bacillus, such as astrain of Bacillus stearothermophilus, in particular a variant of aBacillus stearothermophilus alpha-amylase, such as the one shown in SEQID NO: 3 in WO 99/019467 or SEQ ID NO: 10 herein.

In an embodiment the Bacillus stearothermophilus alpha-amylase has adouble deletion of two amino acids in the region from position 179 to182, more particularly a double deletion at positions I181+G182,R179+G180, G180+I181, R179+I181, or G180+G182, preferably I181+G182, andoptionally a N193F substitution, (using SEQ ID NO: 10 for numbering).

In an embodiment the Bacillus stearothermophilus alpha-amylase has asubstitution at position S242, preferably S242Q substitution.

In an embodiment the Bacillus stearothermophilus alpha-amylase has asubstitution at position E188, preferably E188P substitution.

In an embodiment the alpha-amylase is selected from the group ofBacillus stearothermophilus alpha-amylase variants with the followingmutations:

-   -   I181*+G182*+N193F+E129V+K177L+R179E;    -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 10 for numbering).

In an embodiment the alpha-amylase variant has at least 75% identitypreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, but less than 100% identity tothe polypeptide of SEQ ID NO: 10.

It should be understood that when referring to Bacillusstearothermophilus alpha-amylase and variants thereof they are normallyproduced in truncated form. In particular, the truncation may be so thatthe Bacillus stearothermophilus alpha-amylase shown in SEQ ID NO: 3 inWO 99/19467 or SEQ ID NO: 10 herein, or variants thereof, are truncatedin the C-terminal preferably to have around 490 amino acids, such asfrom 482-493 amino acids. Preferably the Bacillus stearothermophilusvariant alpha-amylase is truncated, preferably after position 484 of SEQID NO: 10, particularly after position 485, particularly after position486, particularly after position 487, particularly after position 488,particularly after position 489, particularly after position 490,particularly after position 491, particularly after position 492, moreparticularly after position 493.

Saccharification may be carried out using conditions well-known in theart with a carbohydrate-source generating enzyme, in particular aglucoamylase, or a beta-amylase and optionally a debranching enzyme,such as an isoamylase or a pullulanase. For instance, a fullsaccharification step may last from about 24 to about 72 hours. However,it is common to do a pre-saccharification of typically 40-90 minutes ata temperature between 30-65° C., typically about 60° C., followed bycomplete saccharification during fermentation in a simultaneoussaccharification and fermentation (SSF) process. Saccharification istypically carried out at a temperature in the range of 20−75° C., e.g.,25-65° C. and 40-70° C., typically around 60° C., and at a pH betweenabout 4 and 5, normally at about pH 4.5.

The saccharification and fermentation steps may be carried out eithersequentially or simultaneously. In an embodiment, saccharification andfermentation are performed simultaneously (referred to as “SSF”).However, it is common to perform a pre-saccharification step for about30 minutes to 2 hours (e.g., 30 to 90 minutes) at a temperature of 30 to65° C., typically around 60° C. which is followed by a completesaccharification during fermentation referred to as simultaneoussaccharification and fermentation (SSF). The pH is usually between4.2-4.8, e.g., pH 4.5. In a simultaneous saccharification andfermentation (SSF) process, there is no holding stage forsaccharification, rather, the yeast and enzymes are added together.

In a typical saccharification process, maltodextrins produced duringliquefaction are converted into dextrose by adding a glucoamylase and adebranching enzyme, such as an isoamylase (U.S. Pat. No. 4,335,208) or apullulanase. The temperature is lowered to 60° C., prior to the additionof the glucoamylase and debranching enzyme. The saccharification processproceeds for 24-72 hours. Prior to addition of the saccharifyingenzymes, the pH is reduced to below 4.5, while maintaining a hightemperature (above 95° C.), to inactivate the liquefying alpha-amylase.This process reduces the formation of short oligosaccharide called“panose precursors,” which cannot be hydrolyzed properly by thedebranching enzyme. Normally, about 0.2-0.5% of the saccharificationproduct is the branched trisaccharide panose (Glc pα1-6Glc pal-4Glc),which cannot be degraded by a pullulanase. If active amylase from theliquefaction remains present during saccharification (i.e., nodenaturing), the amount of panose can be as high as 1-2%, which ishighly undesirable since it lowers the saccharification yieldsignificantly.

Other fermentation products may be fermented at conditions andtemperatures well known to persons skilled in the art, suitable for thefermenting organism in question.

The fermentation product may be recovered by methods well known in theart, e.g., by distillation. Examples of carbohydrate-source generatingenzymes are disclosed in the “Enzymes” section below.

In a particular embodiment, the process of the invention furthercomprises, prior to the conversion of a starch-containing material tosugars/dextrins the steps of:

(x) reducing the particle size of the starch-containing material; and

(y) forming a slurry comprising the starch-containing material andwater.

In an embodiment, the starch-containing material is milled to reduce theparticle size. In an embodiment the particle size is reduced to between0.05-3.0 mm, preferably 0.1-0.5 mm, or so that at least 30%, preferablyat least 50%, more preferably at least 70%, even more preferably atleast 90% of the starch-containing material fits through a sieve with a0.05-3.0 mm screen, preferably 0.1-0.5 mm screen.

The aqueous slurry may contain from 10-55 wt. % dry solids (DS),preferably 25-45 wt. % dry solids (DS), more preferably 30-40 wt. % drysolids (DS) of starch-containing material.

Conventional starch-conversion processes, such as liquefaction andsaccharification processes are described, e.g., in U.S. Pat. No.3,912,590, EP 252730 and EP 063909, which are incorporated herein byreference.

In an embodiment, the conversion process degrading starch to lowermolecular weight carbohydrate components such as sugars or fat replacersincludes a debranching step.

In the case of converting starch into a sugar, the starch isdepolymerized. Such a depolymerization process consists of, e.g., apre-treatment step and two or three consecutive process steps, i.e., aliquefaction process, a saccharification process, and depending on thedesired end-product, an optional isomerization process.

When the desired final sugar product is, e.g., high fructose syrup thedextrose syrup may be converted into fructose. After thesaccharification process, the pH is increased to a value in the range of6-8, e.g., pH 7.5, and the calcium is removed by ion exchange. Thedextrose syrup is then converted into high fructose syrup using, e.g.,an immobilized glucose isomerase.

Production of Fermentation Products

Fermentable sugars (e.g., dextrins, monosaccharides, particularlyglucose) are produced from enzymatic saccharification. These fermentablesugars may be further purified and/or converted to useful sugarproducts. In addition, the sugars may be used as a fermentationfeedstock in a microbial fermentation process for producingend-products, such as alcohol (e.g., ethanol, and butanol), organicacids (e.g., succinic acid, 3-HP and lactic acid), sugar alcohols (e.g.,glycerol), ascorbic acid intermediates (e.g., gluconate,2-keto-D-gluconate, 2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid),amino acids (e.g., lysine), proteins (e.g., antibodies and fragmentthereof).

In an embodiment, the fermentable sugars obtained during theliquefaction process steps are used to produce alcohol and particularlyethanol. In ethanol production, an SSF process is commonly used whereinthe saccharifying enzymes and fermenting organisms (e.g., yeast) areadded together and then carried out at a temperature of 30-40° C.

The organism used in fermentation will depend on the desiredend-product. Typically, if ethanol is the desired end product yeast willbe used as the fermenting organism. In some preferred embodiments, theethanol-producing microorganism is a yeast and specificallySaccharomyces such as strains of S. cerevisiae (U.S. Pat. No.4,316,956). A variety of S. cerevisiae are commercially available andthese include but are not limited to FALI (Fleischmann's Yeast),SUPERSTART (Alltech), FERMIOL (DSM Specialties), RED STAR (Lesaffre) andAngel alcohol yeast (Angel Yeast Company, China). The amount of starteryeast employed in the methods is an amount effective to produce acommercially significant amount of ethanol in a suitable amount of time,(e.g., to produce at least 10% ethanol from a substrate having between25-40% DS in less than 72 hours). Yeast cells are generally supplied inamounts of about 10⁴ to about 10¹², and preferably from about 10⁷ toabout 10¹⁰ viable yeast count per mL of fermentation broth. After yeastis added to the mash, it is typically subjected to fermentation forabout 24-96 hours, e.g., 35-60 hours. The temperature is between about26-34° C., typically at about 32° C., and the pH is from pH 3-6, e.g.,around pH 4-5.

The fermentation may include, in addition to a fermenting microorganisms(e.g., yeast), nutrients, and additional enzymes, including phytases.The use of yeast in fermentation is well known in the art.

In further embodiments, use of appropriate fermenting microorganisms, asis known in the art, can result in fermentation end product including,e.g., glycerol, 1,3-propanediol, gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lacticacid, amino acids, and derivatives thereof. More specifically whenlactic acid is the desired end product, a Lactobacillus sp. (L. casei)may be used; when glycerol or 1,3-propanediol are the desiredend-products E. coli may be used; and when 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid are the desired endproducts, Pantoea citrea may be used as the fermenting microorganism.The above enumerated list are only examples and one skilled in the artwill be aware of a number of fermenting microorganisms that may be usedto obtain a desired end product.

Processes for Producing Fermentation Products from Un-GelatinizedStarch-Containing Material

The invention relates to processes for producing fermentation productsfrom starch-containing material without gelatinization (i.e., withoutcooking) of the starch-containing material (often referred to as a “rawstarch hydrolysis” process). The fermentation product, such as ethanol,can be produced without liquefying the aqueous slurry containing thestarch-containing material and water. In one embodiment a process of theinvention includes saccharifying (e.g., milled) starch-containingmaterial, e.g., granular starch, below the initial gelatinizationtemperature, preferably in the presence of alpha-amylase and/orcarbohydrate-source generating enzyme(s) to produce sugars that can befermented into the fermentation product by a suitable fermentingorganism. In this embodiment the desired fermentation product, e.g.,ethanol, is produced from un-gelatinized (i.e., uncooked), preferablymilled, cereal grains, such as corn.

Accordingly, in one aspect the invention relates to processes forproducing a fermentation product from starch-containing materialcomprising simultaneously saccharifying and fermenting starch-containingmaterial using a carbohydrate-source generating enzymes and a fermentingorganism at a temperature below the initial gelatinization temperatureof said starch-containing material in the presence of a variant proteaseof the invention. Saccharification and fermentation may also beseparate. Thus in another aspect the invention relates to processes ofproducing fermentation products, comprising the following steps:

(i) saccharifying a starch-containing material at a temperature belowthe initial gelatinization temperature using a carbohydrate-sourcegenerating enzyme, e.g., a glucoamylase; and

(ii) fermenting using a fermentation organism;

wherein step (i) is carried out using at least a glucoamylase, and avariant protease of the invention.

In one embodiment the fermenting organism expresses the variant proteaseof the invention.

In one embodiment, an alpha amylase is also added in step (i). Steps (i)and (ii) may be performed simultaneously.

The fermentation product, e.g., ethanol, may optionally be recoveredafter fermentation, e.g., by distillation. Typically amylase(s), such asglucoamylase(s) and/or other carbohydrate-source generating enzymes,and/or alpha-amylase(s), is(are) present during fermentation. Examplesof glucoamylases and other carbohydrate-source generating enzymesinclude raw starch hydrolyzing glucoamylases. Examples ofalpha-amylase(s) include acid alpha-amylases such as acid fungalalpha-amylases. Examples of fermenting organisms include yeast, e.g., astrain of Saccharomyces cerevisiae. The term “initial gelatinizationtemperature” means the lowest temperature at which starch gelatinizationcommences. In general, starch heated in water begins to gelatinizebetween about 50° C. and 75° C.; the exact temperature of gelatinizationdepends on the specific starch and can readily be determined by theskilled artisan. Thus, the initial gelatinization temperature may varyaccording to the plant species, to the particular variety of the plantspecies as well as with the growth conditions. In the context of thisinvention the initial gelatinization temperature of a givenstarch-containing material may be determined as the temperature at whichbirefringence is lost in 5% of the starch granules using the methoddescribed by Gorinstein and Lii, 1992, Starch/Starke 44(12): 461-466.Before initiating the process a slurry of starch-containing material,such as granular starch, having 10-55 w/w % dry solids (DS), preferably25-45 w/w % dry solids, more preferably 30-40 w/w % dry solids ofstarch-containing material may be prepared. The slurry may include waterand/or process waters, such as stillage (backset), scrubber water,evaporator condensate or distillate, side-stripper water fromdistillation, or process water from other fermentation product plants.Because the process of the invention is carried out below the initialgelatinization temperature, and thus no significant viscosity increasetakes place, high levels of stillage may be used if desired. In anembodiment the aqueous slurry contains from about 1 to about 70 vol. %,preferably 15-60 vol. %, especially from about 30 to 50 vol. % waterand/or process waters, such as stillage (backset), scrubber water,evaporator condensate or distillate, side-stripper water fromdistillation, or process water from other fermentation product plants,or combinations thereof, or the like. The starch-containing material maybe prepared by reducing the particle size, preferably by dry or wetmilling, to 0.05 to 3.0 mm, preferably 0.1-0.5 mm. After being subjectedto a process of the invention at least 85%, at least 86%, at least 87%,at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or preferably at least 99% of the dry solids in thestarch-containing material are converted into a soluble starchhydrolyzate. A process in this aspect of the invention is conducted at atemperature below the initial gelatinization temperature, which meansthat the temperature typically lies in the range between 30-75° C.,preferably between 45-60° C. In a preferred embodiment the processcarried at a temperature from 25° C. to 40° C., such as from 28° C. to35° C., such as from 30° C. to 34° C., preferably around 32° C. In anembodiment the process is carried out so that the sugar level, such asglucose level, is kept at a low level, such as below 6 w/w %, such asbelow about 3 w/w %, such as below about 2 w/w %, such as below about 1w/w %., such as below about 0.5 w/w %, or below 0.25 w/w %, such asbelow about 0.1 w/w %.

Such low levels of sugar can be accomplished by simply employingadjusted quantities of enzyme and fermenting organism. A skilled personin the art can easily determine which doses/quantities of enzyme andfermenting organism to use. The employed quantities of enzyme andfermenting organism may also be selected to maintain low concentrationsof maltose in the fermentation broth. For instance, the maltose levelmay be kept below about 0.5 w/w %, such as below about 0.2 w/w %. Theprocess of the invention may be carried out at a pH from about 3 and 7,preferably from pH 3.5 to 6, or more preferably from pH 4 to 5. In anembodiment fermentation is ongoing for 6 to 120 hours, in particular 24to 96 hours.

Processes for Producing Fermentation Products from GelatinizedStarch-Containing Material

In this aspect, the invention relates to processes for producingfermentation products, especially ethanol, from starch-containingmaterial, which process includes a liquefaction step and sequentially orsimultaneously performed saccharification and fermentation steps.Consequently, the invention relates to a process for producing afermentation product from starch-containing material comprising thesteps of:

(a) liquefying starch-containing material in the presence of analpha-amylase;

(b) saccharifying the liquefied material obtained in step (a) using acarbohydrate-source generating enzyme;

(c) fermenting using a fermenting organism;

wherein a variant protease of the invention is present during step b)and/or c).

In one embodiment, the fermenting organism expresses the variantprotease of the invention.

The fermentation product, such as especially ethanol, may optionally berecovered after fermentation, e.g., by distillation. The fermentingorganism is preferably yeast, preferably a strain of Saccharomycescerevisiae. In a particular embodiment, the process of the inventionfurther comprises, prior to step (a), the steps of:

x) reducing the particle size of the starch-containing material,preferably by milling (e.g., using a hammer mill);

y) forming a slurry comprising the starch-containing material and water.

In an embodiment, the particle size is smaller than a #7 screen, e.g., a#6 screen. A #7 screen is usually used in conventional prior artprocesses. The aqueous slurry may contain from 10-55, e.g., 25-45 and30-40, w/w % dry solids (DS) of starch-containing material. The slurryis heated to above the gelatinization temperature and an alpha-amylasevariant may be added to initiate liquefaction (thinning). The slurry mayin an embodiment be jet-cooked to further gelatinize the slurry beforebeing subjected to alpha-amylase in step (a). Liquefaction may in anembodiment be carried out as a three-step hot slurry process. The slurryis heated to between 60-95° C., preferably between 70-90° C., such aspreferably between 80-85° C. at pH 4-6, preferably 4.5-5.5, andalpha-amylase variant, optionally together with a pullulanase and/orprotease, preferably metalloprotease, are added to initiate liquefaction(thinning). In an embodiment the slurry may then be jet-cooked at atemperature between 95-140° C., preferably 100-135° C., such as 105-125°C., for about 1-15 minutes, preferably for about 3-10 minutes,especially around about 5 minutes. The slurry is cooled to 60-95° C. andmore alpha-amylase variant and optionally pullulanase variant and/orprotease, preferably metalloprotease, is(are) added to finalizehydrolysis (secondary liquefaction). The liquefaction process is usuallycarried out at pH 4.0-6, in particular at a pH from 4.5 to 5.5.Saccharification step (b) may be carried out using conditions well knownin the art. For instance, a full saccharification process may last up tofrom about 24 to about 72 hours, however, it is common only to do apre-saccharification of typically 40-90 minutes at a temperature between30-65° C., typically about 60° C., followed by complete saccharificationduring fermentation in a simultaneous saccharification and fermentationprocess (SSF process). Saccharification is typically carried out attemperatures from 20-75° C., preferably from 40-70° C., typically around60° C., and at a pH between 4 and 5, normally at about pH 4.5. The mostwidely used process to produce a fermentation product, especiallyethanol, is a simultaneous saccharification and fermentation (SSF)process, in which there is no holding stage for the saccharification,meaning that a fermenting organism, such as yeast, and enzyme(s), may beadded together. SSF may typically be carried out at a temperature from25° C. to 40° C., such as from 28° C. to 35° C., such as from 30° C. to34° C., preferably around about 32° C. In an embodiment fermentation isongoing for 6 to 120 hours, in particular 24 to 96 hours.

Glucoamylase Present and/or Added in Saccharification and/orFermentation

The carbohydrate-source generating enzyme present duringsaccharification may in one embodiment be a glucoamylase. A glucoamylaseis present and/or added in saccharification and/or fermentation,preferably simultaneous saccharification and fermentation (SSF), in aprocess of the invention (i.e., saccharification and fermentation ofungelatinized or gelatinized starch material).

In an embodiment the glucoamylase present and/or added insaccharification and/or fermentation is of fungal origin, preferablyfrom a stain of Aspergillus, preferably A. niger, A. awamori, or A.oryzae; or a strain of Trichoderma, preferably T. reesei; or a strain ofTalaromyces, preferably T. emersonii or a strain of Trametes, preferablyT. cingulata, or a strain of Pycnoporus, preferably P. sanguineus, or astrain of Gloeophyllum, such as G. serpiarium or G. trabeum, or a strainof the Nigrofomes.

In an embodiment the glucoamylase is derived from Trametes, such as astrain of Trametes cingulata, such as the one shown in SEQ ID NO: 4herein.

In an embodiment the glucoamylase is selected from the group consistingof:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 4 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the polypeptide of SEQ ID NO: 4 herein.

In an embodiment the glucoamylase is derived from Talaromyces, such as astrain of Talaromyces emersonii, such as the one shown in SEQ ID NO: 5herein.

In an embodiment the glucoamylase is selected from the group consistingof:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 5 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the polypeptide of SEQ ID NO: 5 herein.

In an embodiment the glucoamylase is derived from a strain of the genusPycnoporus, in particular a strain of Pycnoporus sanguineus described inWO 2011/066576 (SEQ ID NOs 2, 4 or 6), such as the one shown as SEQ IDNO: 4 in WO 2011/066576.

In an embodiment the glucoamylase is selected from the group consistingof:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 6 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at 6 herein.

In an embodiment the glucoamylase is derived from a strain of the genusGloeophyllum, such as a strain of Gloeophyllum sepiarium or Gloeophyllumtrabeum, in particular a strain of Gloeophyllum as described in WO2011/068803 (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16). In a preferredembodiment the glucoamylase is the Gloeophyllum sepiarium shown in SEQID NO: 2 in WO 2011/068803 or SEQ ID NO: 7 herein.

In an embodiment the glucoamylase is derived from Gloeophyllumserpiarium, such as the one shown in SEQ ID NO: 7 herein. In anembodiment the glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 7 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the polypeptide of SEQ ID NO: 7 herein.

In another embodiment the glucoamylase is derived from Gloeophyllumtrabeum such as the one shown in SEQ ID NO: 8 herein. In an embodimentthe glucoamylase is selected from the group consisting of:

(i) a glucoamylase comprising the polypeptide of SEQ ID NO: 8 herein;

(ii) a glucoamylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the polypeptide of SEQ ID NO: 8 herein.

In an embodiment the glucoamylase is derived from a strain of the genusNigrofomes, in particular a strain of Nigrofomes sp. disclosed in WO2012/064351.

Glucoamylases may in an embodiment be added to the saccharificationand/or fermentation in an amount of 0.0001-20 AGU/g DS, preferably0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, such as 0.1-2AGU/g DS.

Commercially available compositions comprising glucoamylase include AMG200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME™ EXCEL and AMG™ E(from Novozymes A/S); OPTIDEX™ 300, GC480, GC417 (from DuPont.);AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™ and G990 ZR(from DuPont).

According to a preferred embodiment of the invention the glucoamylase ispresent and/or added in saccharification and/or fermentation incombination with an alpha-amylase. Examples of suitable alpha-amylaseare described below.

Alpha-Amylase Present and/or Added in Saccharification and/orFermentation

In an embodiment an alpha-amylase is present and/or added insaccharification and/or fermentation in the processes of the invention.In a preferred embodiment the alpha-amylase is of fungal or bacterialorigin. In a preferred embodiment the alpha-amylase is a fungal acidstable alpha-amylase. A fungal acid stable alpha-amylase is analpha-amylase that has activity in the pH range of 3.0 to 7.0 andpreferably in the pH range from 3.5 to 6.5, including activity at a pHof about 4.0, 4.5, 5.0, 5.5, and 6.0.

In a preferred embodiment the alpha-amylase present and/or added insaccharification and/or fermentation is derived from a strain of thegenus Rhizomucor, preferably a strain the Rhizomucor pusillus, such asone shown in SEQ ID NO: 3 in WO 2013/006756, such as a Rhizomucorpusillus alpha-amylase hybrid having an Aspergillus niger linker andstarch-binding domain, such as the one shown in SEQ ID NO: 9 herein, ora variant thereof.

In an embodiment the alpha-amylase present and/or added insaccharification and/or fermentation is selected from the groupconsisting of:

(i) an alpha-amylase comprising the polypeptide of SEQ ID NO: 9 herein;

(ii) an alpha-amylase comprising an amino acid sequence having at least60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the polypeptide of SEQ ID NO: 9 herein.

In a preferred embodiment the alpha-amylase is a variant of thealpha-amylase shown in SEQ ID NO: 9 having at least one of the followingsubstitutions or combinations of substitutions: D165M; Y141W; Y141R;K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W;G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R;Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N;Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C;Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C;G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C (using SEQ IDNO: 9 for numbering).

In an embodiment the alpha-amylase is derived from a Rhizomucor pusilluswith an Aspergillus niger glucoamylase linker and starch-binding domain(SBD), preferably disclosed as SEQ ID NO: 9 herein, preferably havingone or more of the following substitutions: G128D, D143N, preferablyG128D+D143N (using SEQ ID NO: 9 for numbering), and wherein thealpha-amylase variant present and/or added in saccharification and/orfermentation has at least 75% identity preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, but less than 100% identity to the polypeptide of SEQ ID NO:9 herein.

In a preferred embodiment the ratio between glucoamylase andalpha-amylase present and/or added during saccharification and/orfermentation may preferably be in the range from 500:1 to 1:1, such asfrom 250:1 to 1:1, such as from 100:1 to 1:1, such as from 100:2 to100:50, such as from 100:3 to 100:70.

Starch-Containing Materials

Any suitable starch-containing starting material may be used in aprocess of the present invention. The starting material is generallyselected based on the desired fermentation product. Examples ofstarch-containing starting materials, suitable for use in the processesof the present invention, include barley, beans, cassava, cereals, corn,milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca,wheat, and whole grains, or any mixture thereof. The starch-containingmaterial may also be a waxy or non-waxy type of corn and barley. In apreferred embodiment the starch-containing material is corn. In apreferred embodiment the starch-containing material is wheat.

Fermentation Products

The term “fermentation product” means a product produced by a method orprocess including fermenting using a fermenting organism. Fermentationproducts include alcohols (e.g., ethanol, methanol, butanol); organicacids (e.g., citric acid, acetic acid, itaconic acid, lactic acid,succinic acid, gluconic acid); ketones (e.g., acetone); amino acids(e.g., glutamic acid); gases (e.g., H₂ and CO₂); antibiotics (e.g.,penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B₁₂,beta-carotene); and hormones. In a preferred embodiment the fermentationproduct is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potableneutral spirits; or industrial ethanol or products used in theconsumable alcohol industry (e.g., beer and wine), dairy industry (e.g.,fermented dairy products), leather industry and tobacco industry.Preferred beer types comprise ales, stouts, porters, lagers, bitters,malt liquors, happoushu, high-alcohol beer, low-alcohol beer,low-calorie beer or light beer. In an preferred embodiment thefermentation product is ethanol.

Fermenting Organisms

The term “fermenting organism” refers to any organism, includingbacterial and fungal organisms, such as yeast and filamentous fungi,suitable for producing a desired fermentation product. Suitablefermenting organisms are able to ferment, i.e., convert, fermentablesugars, such as arabinose, fructose, glucose, maltose, mannose, orxylose, directly or indirectly into the desired fermentation product.

Examples of fermenting organisms include fungal organisms such as yeast.Preferred yeast include strains of Saccharomyces, in particularSaccharomyces cerevisiae or Saccharomyces uvarum; strains of Pichia, inparticular Pichia stipitis such as Pichia stipitis CBS 5773 or Pichiapastoris; strains of Candida, in particular Candida arabinofermentans,Candida boidinii, Candida diddensii, Candida shehatae, Candidasonorensis, Candida tropicalis, or Candida utilis. Other fermentingorganisms include strains of Hansenula, in particular Hansenula anomalaor Hansenula polymorpha; strains of Kluyveromyces, in particularKluyveromyces fragilis or Kluyveromyces marxianus; and strains ofSchizosaccharomyces, in particular Schizosaccharomyces pombe.

Preferred bacterial fermenting organisms include strains of Escherichia,in particular Escherichia coli, strains of Zymomonas, in particularZymomonas mobilis, strains of Zymobacter, in particular Zymobactorpalmae, strains of Klebsiella in particular Klebsiella oxytoca, strainsof Leuconostoc, in particular Leuconostoc mesenteroides, strains ofClostridium, in particular Clostridium butyricum, strains ofEnterobacter, in particular Enterobacter aerogenes, and strains ofThermoanaerobacter, in particular Thermoanaerobacter BG1L1 (Appl.Microbiol. Biotech. 77: 61-86), Thermoanarobacter ethanolicus,Thermoanaerobacter mathranii, or Thermoanaerobacterthermosaccharolyticum. Strains of Lactobacillus are also envisioned asare strains of Corynebacterium glutamicum R, Bacillusthermoglucosidaisus, and Geobacillus thermoglucosidasius.

In an embodiment, the fermenting organism is a C6 sugar fermentingorganism, such as a strain of, e.g., Saccharomyces cerevisiae.

In an embodiment, the fermenting organism is a C5 sugar fermentingorganism, such as a strain of, e.g., Saccharomyces cerevisiae.

In one embodiment, the fermenting organism is added to the fermentationmedium so that the viable fermenting organism, such as yeast, count permL of fermentation medium is in the range from 10⁵ to 10¹², preferablyfrom 10⁷ to 10¹⁰, especially about 5×10⁷.

Yeast is the preferred fermenting organism for ethanol fermentation.Preferred are strains of Saccharomyces, especially strains of thespecies Saccharomyces cerevisiae, preferably strains which are resistanttowards high levels of ethanol, i.e., up to, e.g., about 10, 12, 15 or20 vol. % or more ethanol.

In an embodiment, the C5 utilizing yeast is a Saccharomyces cereviseastrain disclosed in WO 2004/085627.

In an embodiment, the fermenting organism is a C5 eukaryotic microbialcell concerned in WO 2010/074577 (Nedalco).

In an embodiment, the fermenting organism is a transformed C5 eukaryoticcell capable of directly isomerize xylose to xylulose disclosed in US2008/0014620.

In an embodiment, the fermenting organism is a C5 sugar fermentatingcell disclosed in WO 2009/109633.

Commercially available yeast include LNF SA-1, LNF BG-1, LNF PE-2, andLNF CAT-1 (available from LNF Brazil), RED START™ and ETHANOL RED™ yeast(available from Fermentis/Lesaffre, USA), FALI (available fromFleischmann's Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast(available from Ethanol Technology, WI, USA), BIOFERM AFT and XR(available from NABC—North American Bioproducts Corporation, GA, USA),GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL(available from DSM Specialties).

The fermenting organism capable of producing a desired fermentationproduct from fermentable sugars is preferably grown under preciseconditions at a particular growth rate. When the fermenting organism isintroduced into/added to the fermentation medium the inoculatedfermenting organism pass through a number of stages. Initially growthdoes not occur. This period is referred to as the “lag phase” and may beconsidered a period of adaptation. During the next phase referred to asthe “exponential phase” the growth rate gradually increases. After aperiod of maximum growth the rate ceases and the fermenting organismenters “stationary phase”. After a further period of time the fermentingorganism enters the “death phase” where the number of viable cellsdeclines.

Fermentation

The fermentation conditions are determined based on, e.g., the kind ofplant material, the available fermentable sugars, the fermentingorganism(s) and/or the desired fermentation product. One skilled in theart can easily determine suitable fermentation conditions. Thefermentation may be carried out at conventionally used conditions.Preferred fermentation processes are anaerobic processes.

For example, fermentations may be carried out at temperatures as high as75° C., e.g., between 40-70° C., such as between 50-60° C. However,bacteria with a significantly lower temperature optimum down to aroundroom temperature (around 20° C.) are also known. Examples of suitablefermenting organisms can be found in the “Fermenting Organisms” sectionabove.

For ethanol production using yeast, the fermentation may go on for 24 to96 hours, in particular for 35 to 60 hours. In an embodiment thefermentation is carried out at a temperature between 20 to 40° C.,preferably 26 to 34° C., in particular around 32° C. In an embodimentthe pH is from pH 3 to 6, preferably around pH 4 to 5.

Other fermentation products may be fermented at temperatures known tothe skilled person in the art to be suitable for the fermenting organismin question.

Fermentation is typically carried out at a pH in the range between 3 and7, preferably from pH 3.5 to 6, such as around pH 5. Fermentations aretypically ongoing for 6-96 hours.

The processes of the invention may be performed as a batch or as acontinuous process. Fermentations may be conducted in an ultrafiltrationsystem wherein the retentate is held under recirculation in the presenceof solids, water, and the fermenting organism, and wherein the permeateis the desired fermentation product containing liquid. Equallycontemplated are methods/processes conducted in continuous membranereactors with ultrafiltration membranes and where the retentate is heldunder recirculation in presence of solids, water, and the fermentingorganism(s) and where the permeate is the fermentation productcontaining liquid. After fermentation the fermenting organism may beseparated from the fermented slurry and recycled.

Fermentation Medium

The phrase “fermentation media” or “fermentation medium” refers to theenvironment in which fermentation is carried out and comprises thefermentation substrate, that is, the carbohydrate source that ismetabolized by the fermenting organism(s).

The fermentation medium may comprise other nutrients and growthstimulator(s) for the fermenting organism(s). Nutrient and growthstimulators are widely used in the art of fermentation and includenitrogen sources, such as ammonia; vitamins and minerals, orcombinations thereof.

Recovery

Subsequent to fermentation, the fermentation product may be separatedfrom the fermentation medium. The fermentation medium may be distilledto extract the desired fermentation product or the desired fermentationproduct may be extracted from the fermentation medium by micro ormembrane filtration techniques. Alternatively, the fermentation productmay be recovered by stripping. Methods for recovery are well known inthe art.

The present invention is further described in the following numberedparagraphs. Paragraph [1] A protease variant comprising a modificationat one or more positions corresponding to positions 39, 50, 57, 60, 74,81, 84, 109, 110, 111, 115, 117, 124, 128, 142, 145, 146, 154, 182, 183,187, 207, 209, 210, 212, 228, 267, 271, 272, 274, 278, 280, 294, 317,318, 320, 321, 322, 328, 343, 348, 362 or 363 of the polypeptide of SEQID NO: 3, wherein the variant has protease activity and wherein thevariant has at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the mature polypeptide of SEQ IDNO: 3, and wherein the variant has increased thermo-stability comparedto the protease of SEQ ID NO: 3.

Paragraph [2] The variant of paragraph 1, which comprises a modificationwhich is a substitution at a position corresponding to positions 39, 50,57, 60, 74, 81, 84, 109, 110, 111, 115, 117, 124, 128, 142, 145, 146,154, 182, 183, 187, 207, 209, 212, 228, 267, 271, 272, 274, 278, 280,294, 317, 318, 320, 321, 322, 328, 343, 348, 362 or 363 of thepolypeptide of SEQ ID NO: 3, wherein the variant has protease activityand wherein the variant has at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3, wherein the protease is a serine protease belonging to theS53 family and wherein the variant has increased thermo-stabilitycompared to the protease of SEQ ID NO: 3.Paragraph [3] The variant of paragraph 1, which comprises a modificationwhich is a deletion at a position corresponding to position 318 or 210of the polypeptide of SEQ ID NO: 3, wherein the variant has proteaseactivity and wherein the variant has at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100% sequence identity to thepolypeptide of SEQ ID NO: 3, wherein the protease is a serine proteasebelonging to the S53 family and wherein the variant has increasedthermo-stability compared to the protease of SEQ ID NO: 3.Paragraph [4] The variant of any of the preceding paragraphs, whereinthe variant comprises or consists of at least one substitution and/ordeletion selected from the group consisting of I39M, I39R, I39L, I39C,S500, K57R, 560P, 560D, E74W, E81A, E81E, E81K, E81R, I84C, D109N,D109P, D110N, F111P, N115D, N115L, E117D, N124Q, N124L, N124W, G128A,Q142R, Q142W, N145A, N145D, N145E, N145G, N145K, N145Q, N145V, T146A,T146D, T146E, T146W, T146Y, Q154R, Q154V, Q154W, Q154Y, Q182G, Q182R,5183L, S183P, 5187L, Q207R, V209L, E212E, 1228R, D267N, V271C, S272C,S272R, S272V, G274G, G278S, D280N, S294A, S317A, S317G, S317S, S318N,G3200, K321A, K321G, A322S, T328C, K343C, P348A, T362A, A363C, S318* andS210* of the polypeptide of SEQ ID NO: 3, wherein the variant hasprotease activity and wherein the variant has at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%, but less than 100% sequence identityto the polypeptide of SEQ ID NO: 3, wherein the protease is a serineprotease belonging to the S53 family and wherein the variant hasincreased thermo-stability compared to the protease of SEQ ID NO: 3.Paragraph [5] The variant according to any of the paragraphs 1-4,comprising a modification at a position corresponding to position 39,60, 74, 81, 84, 109, 115, 117, 142, 145, 146, 154, 182, 183, 187, 209,210, 212, 228, 267, 272, 280, 294, 317, 318, 348 or 362 of thepolypeptide of SEQ ID NO: 3, wherein the variant has protease activityand wherein the variant has at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3, wherein the protease is a serine protease belonging to theS53 family.Paragraph [6] The variant of paragraph 5, which comprises a modificationwhich is a deletion at a position corresponding to position 318 or 210of the polypeptide of SEQ ID NO: 3 wherein the variant has proteaseactivity and wherein the variant has at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100% sequence identity to thepolypeptide of SEQ ID NO: 3, wherein the protease is a serine proteasebelonging to the S53 family.Paragraph [7] The variant of paragraph 5, which comprises a modificationwhich is a substitution at a position corresponding to positions 39, 60,74 81, 84, 109, 115, 142, 145, 146, 154, 182, 183, 187, 209, 212, 228,267, 272, 280, 294, 317, 348 or 362 of the polypeptide of SEQ ID NO: 3,wherein the variant has protease activity and wherein the variant has atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3, wherein theprotease is a serine protease belonging to the S53 family.Paragraph [8] The variant of any of paragraphs 5-7, wherein the variantcomprises or consists of one or more substitutions and/or deletionsselected from the group consisting of I39M, I39R, I39L, I39C, 560D, I84CN115D, N115L, E117D, N145G, N145Q, N145V, N145D, N145K, N145K, N145A,N145E, 5183L, S183P, D280N, Q182G, Q182R, E81R, E81K, E81E, E81A, I84C,Q154V, Q142W, Q142R, T146A, T146W, T146Y, T146E, T146D, 1228R, D267N,S272V, S272R, E212E, 5294A, T362A, E74W, 5187L, P348A, D109P, S317A,S317G, S317S, S317A, S318* and S210* of the polypeptide of SEQ ID NO: 3,wherein the variant has protease activity and wherein the variant has atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3, and whereinthe increased thermo-stability is increased residual activity measuredafter incubation for 30 min at a temperature in the range from 55 to 60degrees Celsius.Paragraph [9] The variant of any of paragraphs 5-8, wherein the variantcomprises at least one of the following modifications or combination ofmodifications:

-   -   N115L;    -   S183P;    -   D280N;    -   N115D;    -   N115L+Q182G;    -   N115L+Q182R;    -   E81R+S183P;    -   E81K+S183P;    -   S183P+Q154V;    -   S183P+Q142W;    -   Q142R+S183P;    -   S183P+T146A;    -   S183P+T146W;    -   S183P+I228R;    -   S183P+D267N;    -   S183P+S272V;    -   S183P+S272R;    -   T146W+D280N;    -   T146Y+S183P;    -   S183P+E212E;    -   S183P+S294A;    -   S183P+T362A;    -   S183P+S294A;    -   S183P+E74W;    -   S183P+E81E;    -   S183P+E81A;    -   N115L+S183L+S187L;    -   S183L+V209L+S210*;    -   D109P+V209L+S210*;    -   N115D+V209L+S210*;    -   E81R+V209L+S210*;    -   D109P+V209L+S210*;    -   N115D+V209L+S210*;    -   E81R+V209L+S210*;    -   T146W+S183P+D280N;    -   I84C+S183P+S272C;    -   I39M+Q142R+S183P;    -   I39R+Q142R+S183P;    -   I39L+Q142R+S183P;    -   I39C+Q142R+S183P;    -   E117D+Q142R+S183P;    -   S60D+Q142R+S183P;    -   N115L+S183L+S187L+P348A;    -   D109P+S183P+V209L+S210*;    -   N115D+S183P+V209L+S210*;    -   E81R+S183P+V209L+S210*;    -   V209L+S210*+S317A+S318*;    -   Q142R+N145G+T146E+S183P;    -   Q142R+N145Q+T146D+S183P;    -   Q142R+N145V+T146E+S183P;    -   Q142R+N145D+T146E+S183P;    -   Q142R+N145K+T146E+S183P;    -   Q142R+N145A+T146D+S183P;    -   Q142R+N145E+T146E+S183P;    -   N115L+S183L+S187L+V209W+S210*;    -   N115L+S183L+S187L+V209L+S210*;    -   N115L+S183L+S187L+S317G+S318*;    -   N115L+S183L+S187L+S317S+S318*;    -   N115L+S183L+S187L+S317A+S318*;    -   E81R+V209L+S210*+S317A+S318*.        Paragraph [10] The variant of any of paragraphs 5-9, wherein the        increased thermo-stability is increased residual activity        measured after incubation for 30 min at a temperature in the        range from 55 to 60 degrees Celsius.        Paragraph [11] The variant of any of paragraphs 5-10, wherein        the variant has a residual activity of at least 10%,        particularly at least 12%, more particularly at least 15%,        measured after incubation for 30 minutes at 56° C.        Paragraph [12] The variant of paragraphs 5-11, wherein the        variant comprises at least one of the following modification or        combination of modifications:    -   N115L+Q182G;    -   N115D;    -   Q142R+S183P;    -   Q142R+N145G+T146E+S183P;    -   Q142R+N145Q+T146D+S183P;    -   Q142R+N145V+T146E+S183P;    -   Q142R+N145D+T146E+S183P;    -   Q142R+N145K+T146E+S183P;    -   Q142R+N145A+T146D+S183P;    -   I39M+Q142R+S183P;    -   Q142R+N145E+T146E+S183P;    -   I39R+Q142R+S183P;    -   I39L+Q142R+S183P;    -   E117D+Q142R+S183P;    -   S60D+Q142R+S183P;    -   and wherein the variant has residual activity of at least 30%        measured after incubation for 30 minutes at 57° C.        Paragraph [13] The variant of paragraphs 5-12, wherein the        variant comprises at least one of the following modifications or        combination of modifications:    -   Q142R+S183P;    -   Q142R+N145G+T146E+S183P;    -   Q142R+N145Q+T146D+S183P;    -   Q142R+N145V+T146E+S183P;    -   Q142R+N145D+T146E+S183P;    -   Q142R+N145K+T146E+S183P;    -   Q142R+N145A+T146D+S183P;    -   I39M+Q142R+S183P;    -   Q142R+N145E+T146E+S183P;    -   I39R+Q142R+S183P;    -   I39L+Q142R+S183P;    -   E117D+Q142R S183P;    -   Q142R+S183P;    -   S60D+Q142R+S183P;    -   Q142R+S183P; and wherein the variant has residual activity of at        least 70% measured after incubation for 30 minutes at 57° C.        Paragraph [14] The variant of paragraphs 5-13, wherein the        variant comprises at least one of the following modifications or        combination of modifications:    -   Q142R+S183P;    -   I39C+Q142R+S183P;    -   E117D+Q142R+S183P;    -   Q142R+S183P;    -   S60D+Q142R+S183P; and wherein the variant has residual activity        of at least 40% measured after incubation for 30 minutes at 60°        C.        Paragraph [15] The variant of paragraphs 5-14, wherein the        variant comprises at least one of the following modifications or        combination of modifications:    -   Q142R+S183P;    -   I39C+Q142R+S183P; and wherein the variant has residual activity        of at least 40% measured after incubation for 30 minutes at an        elevated temperature of 62° C.        Paragraph [16] The variant according to any of the paragraphs        1-4, comprising a modification at a position corresponding to        position 50, 57, 60, 81, 84, 109, 110, 111, 124, 128, 142, 145,        146, 154, 182, 183, 207, 209, 210, 228, 267, 271, 272, 274, 278,        280, 294, 317, 318, 320, 321, 322, 328, 343, 362, or 363 of the        polypeptide of SEQ ID NO: 3, wherein the variant has protease        activity and wherein the variant has at least 75%, at least 80%,        at least 85%, at least 90%, at least 95%, at least 96%, at least        97%, at least 98%, or at least 99%, but less than 100% sequence        identity to the polypeptide of SEQ ID NO: 3, wherein the        protease is a serine protease belonging to the S53 family.        Paragraph [17] The variant of paragraph 16, which comprises a        modification which is a substitution at a position corresponding        to positions 50, 57, 60, 81, 84, 109, 110, 111, 124, 128, 142,        145, 146, 154, 182, 183, 207, 209, 228, 267, 271, 272, 274, 278,        280, 294, 317, 318, 320, 321, 322, 328, 343, 362, or 363 of the        polypeptide of SEQ ID NO: 3.        Paragraph [18] The variant of paragraph 16, which comprises a        modification which is a deletion at a position corresponding to        position 318 or 210 of the polypeptide of SEQ ID NO: 3.        Paragraph [19] The variant of paragraphs 16-18, wherein the        variant comprises or consists of one or more substitutions        and/or deletions selected from the group consisting of S50C,        K57R, S60P, E81R, I84C, D109P, D109N, D110N, F111P, N124L,        N124W, N124Q, G128A, Q142R, Q142W, N145V, N145D, N145A, T146A,        T146W, T146E, T146D, Q154V, Q154W, Q154,R, Q154Y, Q182G, Q182R,        S183P, S183L, Q207R, V209L, 1228R, D267N, V271C, S272V, S272C,        S272R, G274G, G278S, D280N, S294A, S317A, S318N, G3200, K321G,        K321A, A322S, T328C, K343C, T362A, A363C, S318* and S210* of the        polypeptide of SEQ ID NO: 3, wherein the variant has protease        activity and wherein the variant has at least 75%, at least 80%,        at least 85%, at least 90%, at least 95%, at least 96%, at least        97%, at least 98%, or at least 99%, but less than 100% sequence        identity to the polypeptide of SEQ ID NO: 3.        Paragraph [20] The variant of any of paragraphs 16-19, wherein        the variant comprises at least one of the following        modifications or combination of modifications:    -   S183P;    -   D280N;    -   K57R+S183P;    -   D109P+S183P+V209L+S210*;    -   E81R+S183P+V209L+S210*;    -   E81R+V209L+S210*;    -   Q154V+S183P;    -   Q142W+S183P;    -   Q142R+S183P;    -   T146A+S183P;    -   T146W+S183P;    -   S183P+I228R;    -   S183P+D267N;    -   S183P+S272V;    -   E81R+V209L+S210*+S317A+S318*;    -   S183P+T328C+K343C;    -   S183P+G320C+A363C;    -   T146W+D280N;    -   T146W+S183L D+280N;    -   T146W;    -   T146W+S183P+D280N;    -   T146Y+S183P;    -   S183P+Q207R;    -   S500+S183P+V2710;    -   I84C+S183P+S272C;    -   Q142W+T146W+S183P;    -   Q142W+T146W+S183P+D280N;    -   S183P+S294A;    -   S183P+K321G;    -   S183P+T362A;    -   Q182G;    -   Q142W+T146W+Q182R;    -   S272V;    -   S272R;    -   S60P;    -   D109N+D110N;    -   F111P;    -   G128A;    -   G278S;    -   S318N+K321A+A322S;    -   E81R+T146W;    -   E81R+Q142R+S183P;    -   E81R+Q142W+S183P    -   S183P+G274G;    -   E81R;    -   N124L+Q142R+S183P;    -   N124W+Q142R+S183P;    -   N124Q+Q142R+S183P;    -   Q142R+N145V+T146E+S183P;    -   Q142R+N145D+T146E+S183P;    -   Q142R+N145A+T146D+S183P; and wherein the increased        thermo-stability measured as Td by TSA assay is at least 59° C.        Paragraph [21] The variant of any of paragraphs 16-20, wherein        the variant comprises at least one of the following        modifications or combination of modifications:    -   D280N;    -   D109P+S183P+V209L+S210*;    -   E81R+S183P+V209L+S210*;    -   E81R+V209L+S210*;    -   Q154V+S183P;    -   Q142W+S183P;    -   Q142R+S183P;    -   T146A+S183P;    -   T146W+S183P;    -   S183P+D267N;    -   S183P+S272V;    -   E81R+V209L+S210*+S317A+S318*;    -   T146W+D280N;    -   T146W+S183L+D280N;    -   T146W;    -   T146W+S183P+D280N;    -   T146Y+S183P;    -   S183P+Q207R;    -   S500+S183P+V271C;    -   I84C+S183P+S272C;    -   Q142W+T146W+S183P;    -   Q142W+T146W+S183P+D280N;    -   S183P+S294A;    -   Q142W+T146W+Q182R;    -   S272V;    -   S272R;    -   S60P;    -   E81R+T146W;    -   E81R+Q142R+S183P;    -   E81R+Q142W+S183P;    -   S183P+G274G;    -   E81R;    -   N124L+Q142R+S183P;    -   N124W+Q142R+S183P;    -   N124Q+Q142R+S183P;    -   Q142R+N145V+T146E+S183P;    -   Q142R+N145D+T146E+S183P;    -   Q142R+N145A+T146D+S183P; and wherein the increased        thermo-stability measured as Td by TSA assay is at least 61° C.        Paragraph [22] The variant of any of paragraphs 16-21, wherein        the variant comprises at least one of the following        modifications or combination of modifications:    -   E81R+S183P+V209L+S210*;    -   Q142R+S183P;    -   T146W+D280N;    -   T146W+S183L+D280N;    -   T146W+S183P+D280N;    -   S500+S183P+V271C;    -   I84C+S183P+S272C;    -   Q142W+T146W+S183P+D280N;    -   S272V;    -   E81R+T146W;    -   E81R+Q142R+S183P;    -   N124L+Q142R+S183P;    -   N124W+Q142R+S183P;    -   N124Q+Q142R+S183P;    -   Q142R+N145V+T146E+S183P;    -   Q142R+N145D+T146E+S183P;    -   Q142R+N145A+T146D+S183P; and wherein the increased        thermo-stability measured as Td by TSA assay is at least 63° C.        Paragraph [23] The variant of any of paragraphs 16-22, wherein        the variant comprises at least one of the following        modifications or combination of modifications:    -   Q142R+S183P;    -   S500+S183P+V271C;    -   E81R+Q142R+S183P;    -   N124L+Q142R+S183P;    -   N124Q+Q142R+S183P;    -   Q142R+N145V+T146E+S183P;    -   Q142R+N145D+T146E+S183P; or    -   Q142R+N145A+T146D+S183P; and wherein the increased        thermo-stability measured as Td by TSA assay is at least 65° C.        Paragraph [24] The variant according to any one of the preceding        paragraphs, wherein said variant comprises a substitution at 1,        2, 3, 4, 5, 6, 7, 8, 9, or 10 positions.        Paragraph [25] A polynucleotide encoding the variant of any of        paragraphs 1-24. Paragraph [26] A nucleic acid construct        comprising the polynucleotide of paragraph 25.        Paragraph [27] An expression vector comprising the        polynucleotide of paragraph 25.        Paragraph [28] A recombinant host cell comprising the        polynucleotide of paragraph 25.        Paragraph [29] A method of producing a protease variant of any        of paragraphs 1-24, comprising: cultivating the host cell of        paragraph 28 under conditions suitable for expression of the        variant; and optionally recovering the variant.        Paragraph [30] A composition comprising the variant according to        any one of paragraphs 1-24.        Paragraph [31] The composition of paragraph 30, further        comprising a glucoamylase and optionally a fungal alpha-amylase.        Paragraph [32] A process for producing a fermentation product        from starch-containing material comprising simultaneously        saccharifying and fermenting starch-containing material using a        carbohydrate-source generating enzymes and a fermenting organism        at a temperature below the initial gelatinization temperature of        said starch-containing material in the presence of a variant        protease of any of the paragraphs 1-24.        Paragraph [33] A process for producing a fermentation product        from starch-containing material comprising the steps of: a)        liquefying starch-containing material in the presence of an        alpha-amylase; b) saccharifying the liquefied material obtained        in step (a) using a glucoamylse; c) fermenting using a        fermenting organism; wherein a variant protease of any of the        paragraphs 1-24 is present during step b) and/or c).        Paragraph [34] The process of any of the paragraphs 32-33,        wherein the fermentation product is ethanol and the fermenting        organism is Saccharomyces cerevisiae.        Paragraph [35] The host cell of paragraph 28 expressing the        variants of any of paragraphs 1-24, wherein the host cell is a        yeast cell, particularly a Saccharomyces, such as Saccharomyces        cerevisiae.        Paragraph [36] The process of any of the paragraphs 32-33,        wherein the host cell of paragraph 35, is applied as the        fermenting organism in the fermentation step and the        fermentation product is ethanol.

The present invention is further described by the following examples.

EXAMPLES

Enzymes

Enzymes for DNA manipulations (e.g. restriction endonucleases, ligasesetc.) were obtained from New England Biolabs, Inc. and were usedaccording to the manufacturer's instructions.

Media and Reagents

The following media and reagents were used unless otherwise specified:

Chemicals used for buffers and substrates were commercial products ofanalytical grade. Cove: 342.3 g/L Sucrose, 20 ml/L COVE salt solution,10 mM Acetamide, 30 g/L noble agar. Cove top agar: 342.3 g/L Sucrose, 20ml/L COVE salt solution, 10 mM Acetamide, 10 g/L low melt agarose.Cove-N plates are composed of 30 g sucrose, 20 ml Cove salt solution, 3g NaNO₃, and 30 g noble agar and water to 1 litre. COVE salt solutionare composed of 26 g KCl, 26 g MgSO₄ 7H₂O, 76 g KH₂PO₄ and 50 ml Covetrace metals and water to 1 litre. Trace metal solution for COVE arecomposed of 0.04 g NaB₄O₇ 10H₂O, 0.4 g CuSO₄ 5 H₂O, 1.2 g FeSO₄7H₂O, 1.0g MnSO₄ H₂O, 0.8 g Neutral amylase 11 MoO₂2H₂O, and 10.0 g ZnSO₄ 7H₂Oand water to 1 litre. ¼ YPM composed of 2.5 g yeast extract, 5 g peptonand 5 g maltose (pH 4.5) and water to 1 litre. STC buffer was composedof 0.8 M sorbitol, 25 mM Tris (pH 8), and 25 mM CaCl₂ and water to 1litre. STPC buffer composed of 40% PEG4000 in STC buffer. MLC composedof 40 g Glucose, 50 g Soybean powder, 4 g/Citric acid (pH 5.0) and waterto 1 litre.

Purchased Material (E. coli, Plasmid and Kits)

E. coli DH5-alpha (Toyobo) was used for plasmid construction andamplification. Amplified plasmids were recovered with Qiagen Plasmid Kit(Qiagen). QIAquick™ Gel Extraction Kit (Qiagen) was used for thepurification of PCR fragments and extraction of DNA fragment fromagarose gel.

Strains

The expression host strain Aspergillus niger described is a derivativeof NN059203. NN059203 was isolated by Novozymes and described inWO12160093 and is a derivative of Aspergillus niger NN049184 which wasisolated from soil.

Transformation of Aspergillus

Transformation of Aspergillus species can be achieved using the generalmethods for yeast transformation. The preferred procedure for theinvention is described below. The Aspergillus niger host strain wasinoculated into 100 ml YPG medium supplemented with 10 mM uridine andincubated for 16 hrs at 32° C. at 80 rpm. Pellets were collected andwashed with 0.6 M KCl, and resuspended in 20 ml 0.6 M KCl containing acommercial glucanase product (GLUCANEX™, Novozymes A/S, Bagsvrd,Denmark) at a final concentration of 20 mg per ml. The suspension wasincubated at 32° C. with shaking (80 rpm) until protoplasts were formed,and then washed twice with STC buffer. The protoplasts were counted witha hematometer and resuspended and adjusted in an 8:2:0.1 solution ofSTC:STPC:DMSO to a final concentration of 2.5×10⁷ protoplasts/ml.Approximately 4 pg of plasmid DNA was added to 100 pl of the protoplastsuspension, mixed gently, and incubated on ice for 30 minutes. One ml ofSPTC was added and the protoplast suspension was incubated for 20minutes at 37° C. After the addition of 10 ml of 50° C. Cove topagarose, the reaction was poured onto Cove agar plates and the plateswere incubated at 32° C. for 5 days.

PCR Amplification

PrimeSTAR® HS (Premix) 10 μl

Template DNA (50-100 ng/pl) 1 μl

Forward primer (100 pM) 1 μl

Reverse primer (100 pM) 1 μl

Distilled water to 20 μl

PCR Conditions

1. 94° C. 2 min

2. 94° C. 10 sec

3. 57° C. 5 sec

4. 72° C. 20 sec

Repeat 2-4, 30 cycles

5. 72° C. 30 sec

MTP Cultivation for Enzyme Production

Spores of Aspergillus libraries were inoculated in 0.5-1 ml of ¼YPMmedia in 96 deep well plate and cultivated at 30° C. for 2-3 days at 600rpm.

Enzyme Assay

Zein Plate Assay

Culture supernatants were applied on 0.05-0.1% of zein (Sigma) plate (20mM sodium acetate buffer, pH4.5) and incubated at appropriatetemperatures (30-60 degree C.).

Suc-AAPF-pna Analysis

Culture supernatants pre-incubated at appropriate temperatures (50 to 60degree C. and 4 degree C. as a control) are measured for proteaseactivity by AAPF assay using N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide(SIGMA-ALDRICH).

Assay:

1) Add 25 μl samples to wells of 384 microtiterplate (MTP)

2) Add 25 μl pNA substrate working soln. to 384 MTP

3) Read 405 nm (zero point)

4) Incubate 37° C., 60 min (if the color is not developed well, continueincubation)

5) Read 405 nm (zero point)

DNA Isolation from Aspergillus Clones

Inserted DNAs of Aspergillus strains were recovered by direct PCR methoddescribed below or PCR on the isolated DNA by chromosomal DNApurification kit (FastDNA SPIN Kit for Soil, MP biomedicals, #6560-200)using a primer pair, insert rescue F and R.

insert rescue F (SEQ ID NO: 11) AATCTCAGAACACCAATATC insert rescue R(SEQ ID NO: 12) AACACTATGCGTTATCGTAC

The amplified DNAs were purified by agarose gel electrophoresis followedto QIAquick Gel Extraction kit (Qiagen) for sequencing analysis to checkthe quality of constructed libraries. Colony PCR was carried out asfollowing:

Conidias from strains were transferred to a 1.5 ml tube and 500 μl ofTE-buffer was added and mixed briefly.

It was diluted 10-20 times in water and one μl of the dilution was usedas template for PCR.

Purification

Purification of the variants of Meripilus giganteus serine proteasebelonging to family 53 herein denoted as MgProtIII variants was carriedout by two steps, desalting column and cation exchange chromatographycolumn. Finally, the sample was buffer exchanged and concentrated in 20mM succinate buffer pH 4.0 using a 30 kDa centrifugal concentrator(Sartorius AG).

TSA (Thermal Shift Assay)

Purified enzyme was diluted with 50 mM sodium acetate buffer pH 4.5 to0.75 mg/ml and 10 μl of that were mixed with 15 μl of SYPRO Orange(Invitrogen) diluted with Milli-Q water and 5 μl of 30 mM bacitracinsolution dissolved in 50 mM sodium acetate buffer pH 4.5. Thirtymicroliters of mixture solution was transfer to LightCycler 480Multiwell Plate 96 (Roche Diagnostics) and the plate was sealed.

Equipment Parameters of TSA:

Apparatus: LightCycler 480 Real-Time PCR System (Roche Applied Science)

Scan rate: 0.02° C./sec

Scan range: 37-96° C.

Integration time: 1.0 sec

Excitation wave length 465 nm

Emission wave length 580 nm

The obtained fluorescence signal was normalized into a range of 0 and 1.The Thermal denaturation temperature, Td, was defined as the temperaturewhere the normalized value is closest to 0.5.

Example 1: Library Construction

Plasmid Library Construction Using In-Fusion Cloning (Clontech)

An expression vector, pFLP-MgProIII disclosed in WO1260093 FIG. 5, whichcontains the target protease gene (shown as SEQ 1) instead ofglucoamylase gene and amd S marker gene instead of pyr G marker gene,was digested with appropriate restriction enzymes (XhoI BsiW1 forpFRT-GIAMG) to cut out the protease gene.

Two PCRs were carried out for a library construction using 2 primerpairs, a forward degenerated primer and a primer having more than 15 bpoverlapping with an expression vector (vector F described below), andvector F primer and a reverse primer having 15 bp overlapping with thedegenerated primer using the expression vector as a template.

Vector R 25mer (SEQ ID NO: 13) TAAGTGGAGGGAAAAACACTATGCG Vector F 32mer(SEQ ID NO: 14) GCTTGGAGCAACAATCTCAGAACACCAATATC

One of the examples of primers for a library is shown below:

F111X F 27mer (SEQ ID NO: 15) ATCTCCGTCGGCGACGACNNKCAGGAT F111X R 20mer(SEQ ID NO: 16) GTCGCCGACGGAGATGAACG

The digested vector and PCR fragments were mixed with In-Fusion mix andtransformed into E. coli DH5alpha. Obtained E. coli transformants werepooled and plasmids were extracted to use for Aspergillus libraryconstruction.

Aspergillus Transformation to Construct a Library in Aspergillus

One μg of each plasmid library was transformed into A. niger hoststrain. Aspergillus transformants were isolated in a 96 well-MTPcontaining COVE-N gly agar (100 ul/well), cultivate at 32° C. for 1 weekto have enough sporulation. 100 μl/well of 0.01% tween 20 was added tothe each well, suspended with spores and the suspension was inoculatedin a 96 well-MTP containing YPG and cultivated for 3 days at 30° C. withshaking to have Aspergillus culture library. They were used for furtherlibrary screening works.

Library Screening

Constructed Aspergillus libraries were cultivated in 96 well MTP and theculture supernatants were spotted on zein plates at appropriatetemperatures. Positive variants were tested by Suc-AAPF-pna analysis andvariants having higher residual activities were identified. Positivevariants were cultivated in shake flasks and samples were used forfurther purification and characterization.

DNA Isolation from Aspergillus Clones

Inserted DNAs of Aspergillus strains were recovered by direct PCR methoddescribed below or PCR on the isolated DNA by chromosomal DNApurification kit (FastDNA SPIN Kit for Soil, MP biomedicals, #6560-200)using a primer pair, insert rescue F and R.

insert rescue F (SEQ ID NO: 11) AATCTCAGAACACCAATATC insert rescue R(SEQ ID NO: 12) AACACTATGCGTTATCGTAC

The amplified DNAs were purified by agarose gel electrophoresis followedto QIAquick Gel Extraction kit (Qiagen) for sequencing analysis to checkthe quality of constructed libraries.

Results

Table 1 lists the positive variants identified by Suc-AAPF-pna analysis.Samples were incubated at certain temperatures for 30 minutes and theirremaining activities were measured by AAPF assay. The residualactivities in tables below are described as relative activity to onesincubated at 4 degree C.

<Residual activity (%)> JMgP ID Modification 57° C., 30 min 58° C., 30min WT — 8 8 JMgP006 N115L 10 9 JMgP019 N115L S183L S187L 18 15 JMgP033N115L Q182G 36 4 JMgP076 N115D 45 4 JMgP071 N115L S183L S187L P348A 2211 JMgP ID — 55° C., 30 min 56° C., 30 min WT — 19 4 JMgP009 S183P 31 10JMgP033 N115L Q182G 38 17 JMgP034 N115L Q182R 60 35 JMgP058 N115L S183LS187L V209W S210* 47 19 JMgP059 N115L S183L S187L V209L S210* 39 20JMgP064 N115L S183L S187L S317G S318* 51 37 JMgP065 N115L S183L S187LS317S S318* 47 22 JMgP066 N115L S183L S187L S317A S318* 47 33 JMgP075S183L V209L S210* 49 41 JMgP ID 55° C., 30 min 56° C., 30 min JMgP009S183P 39.4 19 Iib19-4 S183P E74W 32.1 13.7 Iib22-2 S183P E81A 46.1 23.5JMgP083 E81R S183P 70.9 53.5 JMgP084 E81K S183P 73.5 55.6 Iib22-11 S183PE81E 36.8 15.3 JMgP009 S183P 46.8 24.7 JMgP094 S183P Q154V 67.4 49.4JMgP095 S183P Q142W 66.1 50.6 JMgP096 Q142R S183P 99 100 JMgP097 S183PT146A 65.8 47.4 JMgP098 S183P T146W 83.8 78.1 JMgP099 S183P I228R 6749.5 JMgP100 S183P D267N 81.9 76.6 JMgP101 S183P S272V 81.8 77.5 JMgP103S183P S272R 88.6 83.2 JMgP120 T146Y S183P 64 52 JMgP009 S183P 48 27JMgP030 D280N 62 49 JMgP087 D109P S183P V209L S210* 63 49 JMgP089 E81RS183P V209L S210* 69 62 JMgP091 D109P V209L S210* 47 28 JMgP092 N115DV209L S210* 54 36 JMgP093 E81R V209L S210* 56 42 JMgP104 V209L S210*S317A S318* 58 45 JMgP106 E81R V209L S210* S317A S318* 65 55 JMgP115T146W D280N 60 50 JMgP118 T146W S183P D280N 73 29 JMgP120 T146Y S183P 8137 JMgP134 S183P S294A 85 58 JMgP137 S183P T362A 53 32 JMgP140 S183PS294A 73 56 55° C., 30 min 56° C., 30 min JMgP009 S183P 50 29 JMgP088N115D S183P V209L S210* 41 34 JMgP096 Q142R S183P 73 71 JMgP123 S183PE212E 48 32 JMgP127 I84C S183P S272C 71 67 57° C., 30 min 58° C., 30 minJMgP096 Q142R S183P 78 76 JMgP229 Q142R N145G T146E S183P 86 87 JMgP230Q142R N145Q T146D S183P 92 92 JMgP231 Q142R N145V T146E S183P 92 91JMgP232 Q142R N145D T146E S183P 89 90 JMgP233 Q142R N145K T146E S183P 9294 JMgP234 Q142R N145A T146D S183P 84 82 JMgP236 I39M Q142R S183P 88 92JMgP235 Q142R N145E T146E S183P 90 87 JMgP237 I39R Q142R S183P 82 82JMgP238 I39L Q142R S183P 91 98 60° C., 30 min 62° C., 30 min JMgP096Q142R S183P 84 45 JMgP245 I39C Q142R S183P 125 105 57° C., 30 min 60°C., 30 min JMgP252 E117D Q142R S183P 90 61 JMgP096 Q142R S183P 88 43Iib81-1 S60D Q142R S183P 96 99 JMgP096 Q142R S183P 96 60

Example 2: Purification and Thermal Shift Assay (TSA) Analysis

Purification

Purification of MgProtIII variants was carried out by two steps,desalting column and cation exchange chromatography column. Finally, thesample was buffer exchanged and concentrated in 20 mM succinate bufferpH 4.0 using a 30 kDa centrifugal concentrator (Sartorius AG).

TSA

Purified enzyme was diluted with 50 mM sodium acetate buffer pH 4.5 to0.75 mg/ml and 10 μl of that were mixed with 15 μl of SYPRO Orange(Invitrogen) diluted with Milli-Q water and 5 μl of 30 mM bacitracinsolution dissolved in 50 mM sodium acetate buffer pH 4.5. Thirtymicroliters of mixture solution was transfer to LightCycler 480Multiwell Plate 96 (Roche Diagnostics) and the plate was sealed.

Equipment Parameters of TSA:

Apparatus: LightCycler 480 Real-Time PCR System (Roche Applied Science)

Scan rate: 0.02° C./sec

Scan range: 37-96° C.

Integration time: 1.0 sec

Excitation wave length 465 nm

Emission wave length 580 nm

The obtained fluorescence signal was normalized into a range of 0 and 1.The Td was defined as the temperature where the normalized value isclosest to 0.5.

Result: The TSA data are listed in TABLE 2.

Sample Modification Td [° C.] MgProtlII (wt) — 58.67 JMgP009 S183P 60.28JMgP030 D280N 61.15 JMgP081 K57R S183P 59.67 JMgP087 D109P S183P V209LS210* 62.37 JMgP089 E81R S183P V209L S210* 63.03 JMgP093 E81R V209LS210* 62.01 JMgP094 Q154V S183P 61.06 JMgP095 Q142W S183P 61.39 JMgP096Q142R S183P 65.77 JMgP097 T146A S183P 61.18 JMgP098 T146W S183P 62.34JMgP099 S183P I228R 60.59 JMgP100 S183P D267N 62.43 JMgP101 S183P S272V61.06 JMgP106 E81R V209L S210* S317A S318* 62.15 JMgP108 S183P T328CK343C 60.59 JMgP110 S183P G320C A363C 59.71 JMgP115 T146W D280N 63.33JMgP116 T146W S183L D280N 63.27 JMgP117 T146W 62.46 JMgP118 T146W S183PD280N 63.72 JMgP120 T146Y S183P 62.13 JMgP122 S183P Q207R 61.36 JMgP126S50C S183P V271C 66.92 JMgP127 I84C S183P S272C 64.44 JMgP130 Q142WT146W S183P 61.94 JMgP132 Q142W T146W S183P D280N 63.00 JMgP134 S183PS294A 61.61 JMgP136 S183P K321G 60.63 JMgP137 S183P T362A 60.87 JMgP141Q182G 59.88 JMgP144 Q142W T146W Q182R 62.13 JMgP147 S272V 63.18 JMgP148S272R 61.67 JMgP157 S60P 61.74 JMgP167 D109N D110N 60.64 JMgP173 F111P59.43 JMgP175 G128A 59.95 JMgP203 G278S 59.95 JMgP206 S318N K321A A322S60.23 JMgP214 E81R T146W 64.02 JMgP215 E81R Q142R S183P 66.08 JMgP216E81R Q142W S183P 62.81 JMgP218 S183P G274G 61.03 JMgP220 E81R 62.34JMgP223 N124L Q142R S183P 65.69 JMgP224 N124W Q142R S183P 64.09 JMgP225N124Q Q142R S183P 65.38 JMgP231 Q142R N145V T146E S183P 65.79 JMgP232Q142R N145D T146E S183P 65.37 JMgP234 Q142R N145A T146D S183P 65.52

The invention claimed is:
 1. A polypeptide comprising a protease variantcomprising a modification at position Q142 of the polypeptide of SEQ IDNO: 3, wherein the variant has protease activity and wherein the varianthas at least 90%, but less than 100%, sequence identity to the maturepolypeptide of SEQ ID NO: 3, and wherein the variant has increasedthermo-stability compared to the protease of SEQ ID NO:
 3. 2. Thevariant of claim 1, wherein the variant comprises of at least onesubstitution and/or deletion selected from the group consisting of I39M,I39R, I39L, I39C, S50C, K57R, S60P, S60D, E74W, E81A, E81E, E81K, E81R,I84C, D109N, D109P, D110N, F111P, N115D, N115L, E117D, N124Q, N124L,N124W, G128A, Q142R, Q142W, N145A, N145D, N145E, N145G, N145K, N145Q,N145V, T146A, T146D, T146E, T146W, T146Y, Q154R, Q154V, Q154W, Q154Y,Q182G, Q182R, S183L, S183P, S187L, Q207R, V209L, E212E, I228R, D267N,V271C, S272C, S272R, S272V, G274G, G278S, D280N, S294A, S317A, S317G,S317S, S318N, G320C, K321A, K321G, A322S,T328C, K343C, P348A, T362A,A363C, S318* and S210* of the polypeptide of SEQ ID NO: 3, wherein thevariant has protease activity and wherein the variant has at least 90%,but less than 100%, sequence identity to the polypeptide of SEQ ID NO:3, wherein the protease is a serine protease belonging to the S53 familyand wherein the variant has increased thermo-stability compared to theprotease of SEQ ID NO:
 3. 3. The variant according to claim 1,comprising a modification at a position corresponding to position 39,60, 74, 81, 84, 109, 115, 117, 145, 146, 154, 182, 183, 187, 209, 210,212, 228, 267, 272, 280, 294, 317, 318, 348 or 362 of the polypeptide ofSEQ ID NO: 3, wherein the variant has protease activity and wherein thevariant has at least 90%, but less than 100%, sequence identity to thepolypeptide of SEQ ID NO: 3, wherein the protease is a serine proteasebelonging to the S53 family.
 4. The variant of claim 3, wherein thevariant comprises or consists of one or more substitutions and/ordeletions selected from the group consisting of I39M, I39R, I39L, I39C,S60D, I84C N115D, N115L, E117D, N145G, N145Q, N145V, N145D, N145K,N145K, N145A, N145E, S183L, S183P, D280N, Q182G, Q182R, E81R, E81K,E81E, E81A, I84C, Q154V, Q142W, Q142R, T146A, T146W, T146Y, T146E,T146D, I228R, D267N, S272V, S272R, E212E, S294A, T362A, E74W, S187L,P348A, D109P, S317A, S317G, S317S, S317A, S318* and S210* of thepolypeptide of SEQ ID NO: 3, wherein the variant has protease activityand wherein the variant has at least 90%, but less than 100%, sequenceidentity to the polypeptide of SEQ ID NO: 3, and wherein the increasedthermo-stability is increased residual activity measured afterincubation for 30 min at a temperature in the range from 55 to 60degrees Celsius.
 5. The variant of claim 3, wherein the variantcomprises at least one of the following modifications or combination ofmodifications: N115L; S183P; D280N; N115D; N115L+Q182G; N115L+Q182R;E81R+S183P; E81K+S183P; S183P+Q154V; S183P+Q142W; Q142R+S183P;S183P+T146A; S183P+T146W; S183P+I228R; S183P+D267N; S183P+S272V;S183P+S272R; T146W+D280N; T146Y+S183P; S183P+E212E; S183P+S294A;S183P+T362A; S183P+S294A; S183P+E74W; S183P+E81E; S183P+E81A;N115L+S183L+S187L; S183L+V209L+S210*; D109P+V209L+S210*; N115D+V209L+S210*; E81R+V209L+S210*; D109P+V209L+S210*;N115D+V209L+S210*; E81R+V209L+S210*; T146W+S183P+D280N;I84C+S183P+S272C; I39M+Q142R+S183P; I39R+Q142R+S183P; I39L+Q142R+S183P;I39C+Q142R+S183P; E117D+Q142R+S183P; S60D+Q142R+S183P;N115L+S183L+S187L+P348A; D109P+S183P+V209L+S210*;N115D+S183P+V209L+S210*; E81R+S183P+V209L+S210*;V209L+S210*+S317A+S318*; Q142R+N145G+T146E+S183P;Q142R+N145Q+T146D+S183P; Q142R+N145V+T146E+S183P;Q142R+N145D+T146E+S183P; Q142R+N145K+T146E+S183P;Q142R+N145A+T146D+S183P; Q142R+N145E+T146E+S183P;N115L+S183L+S187L+V209W+S210*; N115L+S183L+S187L+V209L+S210*;N115L+S183L+S187L+S317G+S318*; N115L+S183L+S187L+S317S+S318*;N115L+S183L+S187L+S317A+S318*; E81R+V209L+S210*+S317A+S318*.
 6. Thevariant of claim 3, wherein the increased thermo-stability is increasedresidual activity measured after incubation for 30 min at a temperaturein the range from 55 to 60 degrees Celsius.
 7. The variant of claim 3,wherein the variant has a residual activity of at least 10%, measuredafter incubation for 30 minutes at 56° C.
 8. The variant according toclaim 1, comprising a modification at a position corresponding toposition 50, 57, 60, 81, 84, 109, 110, 111, 124, 128, 145, 146, 154,182, 183, 207, 209, 210, 228, 267, 271, 272, 274, 278, 280, 294, 317,318, 320, 321, 322, 328, 343, 362, or 363 of the polypeptide of SEQ IDNO: 3, wherein the variant has protease activity and wherein the varianthas at least 90%, but less than 100%, sequence identity to thepolypeptide of SEQ ID NO: 3, wherein the protease is a serine proteasebelonging to the S53 family.
 9. The variant of claim 8, wherein thevariant comprises of one or more substitutions and/or deletions selectedfrom the group consisting of S50C, K57R, S60P, E81R, I84C, D109P, D109N,D110N, F111P, N124L, N124W, N124Q, G128A, Q142R, Q142W, N145V, N145D,N145A, T146A, T146W, T146E, T146D, Q154V, Q154W, Q154,R, Q154Y, Q182G,Q182R, S183P, S183L, Q207R, V209L, I228R, D267N, V271C, S272V, S272C,S272R, G274G, G278S, D280N, S294A, S317A, S318N, G320C, K321G, K321A,A322S, T328C, K343C, T362A, A363C, S318* and S210* of the polypeptide ofSEQ ID NO: 3, wherein the variant has protease activity and wherein thevariant has at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3.10. The variant of claim 8, wherein the variant comprises at least oneof the following modifications or combination of modifications: S183P;D280N; K57R+S183P; D109P+S183P+V209L+S210*; E81R+S183P+V209L+S210*;E81R+V209L+S210*; Q154V+S183P; Q142W+S183P; Q142R+S183P; T146A+S183P;T146W+S183P; S183P+I228R; S183P+D267N; S183P+S272V;E81R+V209L+S210*+S317A+S318*; S183P+T328C+K343C; S183P+G320C+A363C;T146W+D280N; T146W+S183L D+280N; T146W; T146W+S183P+D280N; T146Y+S183P;S183P+Q207R; S50C+S183P+V271C; I84C+S183P+S272C; Q142W+T146W+S183P;Q142W+T146W+S183P+D280N; S183P+S294A; S183P+K321G; S183P+T362A; Q182G;Q142W+T146W+Q182R; S272V; S272R; S60P; D109N+D110N; F111P; G128A; G278S;S318N+K321A+A322S; E81R+T146W; E81R+Q142R+S183P; E81R+Q142W+S183PS183P+G274G; E81R; N124L+Q142R+S183P; N124W+Q142R+S183P;N124Q+Q142R+S183P; Q142R+N145V+T146E+S183P; Q142R+N145D+T146E+S183P;Q142R+N145A+T146D+S183P; and wherein the increased thermo-stabilitymeasured as Td by TSA assay is at least 59° C.
 11. An polynucleotideencoding the polypeptide of claim
 1. 12. A nucleic acid constructcomprising the polynucleotide of claim
 11. 13. An expression vectorcomprising the polynucleotide of claim
 11. 14. A recombinant host cellcomprising the polynucleotide of claim
 11. 15. A method of producing apolypeptide comprising a protease variant, comprising: cultivating thehost cell of claim 14 under conditions suitable for expression of thevariant; and optionally recovering the variant.
 16. A compositioncomprising the polypeptide according to claim
 1. 17. The composition ofclaim 16, further comprising a glucoamylase and optionally a fungalalpha-amylase.
 18. A process for producing a fermentation product fromstarch-containing material comprising simultaneously saccharifying andfermenting starch-containing material using a carbohydrate-sourcegenerating enzymes and a fermenting organism at a temperature below theinitial gelatinization temperature of said starch-containing material inthe presence of the polypeptide of claim
 1. 19. A process for producinga fermentation product from starch-containing material comprising thesteps of: a) liquefying starch-containing material in the presence of analpha-amylase; b) saccharifying the liquefied material obtained in step(a) using a glucoamylse; c) fermenting using a fermenting organism;wherein the polypeptide of claim 1 is present during step b) and/or c).20. The process of claim 18, wherein the fermentation product is ethanoland the fermenting organism is Saccharomyces cerevisiae.
 21. The hostcell of claim 14, wherein the host cell is a yeast cell.
 22. The processof claim 18, wherein a host yeast cell expressing the polypetide, isapplied as the fermenting organism in the fermentation step and thefermentation product is ethanol.